Theriogenology - Articles in Press

Serum trace mineral variations in Nili-Ravi buffaloes suffering with prepartum vaginal prolapse in two different agro-ecological zones of Punjab, Pakistan - Corrected Proof

M.S. Akhtar, L.A. Lodhi, I. Ahmad, Z.I. Qureshi, G. Muhammad — 2012-01-30

Abstract: The present study was conducted during 2005 and 2006 on 200 Nili-Ravi buffaloes kept in two agroecological zones (irrigated [zone 1] and rain-fed [zone-2]) of Punjab, Pakistan, with the objective to determine the level of trace minerals (Cu, Fe, Zn, Se) in serum of the buffaloes suffering from vaginal prolapse and to compare them with their healthy counterparts. In each zone 50 buffaloes suffering from prepartum vaginal prolapse during their seventh month of gestation were identified through survey. Vaginal prolapse-affected buffaloes belonging to zone 1 were identified as group VPB1 (N = 50), whereas buffaloes belonging to zone 2 were recognized as VPB2 (N = 50). The buffaloes of control group in zone 1 and zone 2 were identified as NCB1 and NCB2, respectively. The blood samples in all four groups of buffaloes were collected three times, i.e., first when these animals were in the eighth month of gestation, second during the eighth to ninth month of gestation, and finally when these animals were in the ninth or later month of gestation. The mean serum copper concentrations in buffaloes of group VPB1 were significantly lower (P < 0.05) in comparison with NCB1 and NCB2, whereas there were nonsignificant differences (P > 0.05) in copper concentrations between VPB1 and VPB2. There was a significant difference (P < 0.05) of iron concentration in VPB1 compared with NCB1 and NCB2. Similarly, VPB2 also had significantly lower (P < 0.05) iron concentrations compared with NCB1 and NCB2. Serum zinc concentrations were significantly lower (P < 0.05) in animals of the VPB1 group when compared with NCB1 and NCB2. Similarly, lower zinc concentrations were observed in VPB2 in comparison with NCB1 and NCB2. There was significantly lower (P < 0.05) zinc concentration in affected buffaloes (VPB1 and VPB2) from the ninth month of gestation to term when compared with those in the eighth to ninth mo of gestation, and with those not yet in the eighth month of gestation. Serum selenium concentration were significantly higher (P < 0.05) in control group buffaloes (NCB1 and NCB2) in comparison with vaginal prolapse-affected buffaloes (VPB1 and VPB2). During different stages of gestation, mean serum selenium concentrations varied nonsignificantly (P > 0.05) within each group of buffalo. Based on information obtained from this study, it was concluded that the low serum concentration of copper and selenium are linked to increased incidence of vaginal prolapse in buffaloes during the last trimester of gestation.

cDNA microarray analysis of gene expression in parthenotes and in vitro produced buffalo embryos - Corrected Proof

A.S. Abdoon, N. Ghanem, O.M. Kandil, A. Gad, K. Schellander, D. Tesfye — 2012-01-30

Abstract: The retarded development of parthenote embryo could be due to aberrant epigenetic imprinting, which may disrupt many aspects and lead to conceptus demise. The present work was conducted to: 1) compare the development of in vitro produced (IVP) and parthenogenetically developed (P) buffalo embryos from the 2-cell to blastocyst stage; 2) investigate the global gene expression profile and search for new candidate transcripts differing between IVP and P buffalo blastocyst using cDNA microarray analysis (validated by Real Time PCR); 3) follow the particular expression patterns of PLAC8 and OCT4 genes at five different stages of preimplantation development by Real Time PCR; and 4) study the expression of CDX2 at the blastcocyst stage. Cleavage rate was higher (P < 0.05) in P than IVP buffalo embryos, while, progression to blastocyst and number of cells per blastocyst were lower (P < 0.05) in P than IVP blastocysts. Microarray analysis indicate that 56 differentially expressed genes between the two groups, of which 51 genes (91.07%) were up-regulated, and five genes were downregulated in IVP blastocyst, using 1.4 fold-changes as a cutoff. Differentially expressed genes are related to translation, nucleic acid synthesis, cell adhesion and placentation. Validation of candidate genes revealed that the transcript abundance of PTGS2, RPS27A, TM2D3, PPA1, AlOX15, RPLO and PLAC8 were downregulated (7/8) in parthenote blastocyst compared to the IVP blastocyst. PLAC8 gene expression was higher (P < 0.05) at 2-cell, morula and blastocyst stages in IVP embryos compared with parthenote embryos. The OCT4 gene expression was higher (P < 0.05) in 2-cell, 4-cell, 8-cell and blastocysts produced in vitro. In conclusion, the retarded development of parthenogenetic buffalo embryos could be due to downregulation of genes related to translation, nucleic acid synthesis, cell adhesion, and placental development. The low expression of PLAC8 and OCT4 during the different stages of development may be responsible, in part, to the failure of development of parthenote buffalo embryos.

The value of microscopic semen motility assessment at collection for a commercial artificial insemination center, a retrospective study on factors explaining variation in pig fertility - Corrected Proof

M.L.W.J. Broekhuijse, E. Šoštarić, H. Feitsma, B.M. Gadella — 2012-01-30

Abstract: This study was conducted to evaluate the relationship between boar and semen related parameters and the variation in field fertility results. In 8 years time semen insemination doses from 110 186 ejaculates of 7429 boars were merged to fertility parameters of inseminations of 165 000 sows and these records were used for analysis. From all ejaculates boar and semen related data were recorded at the artificial insemination (AI) centers. Fertility parameters, such as farrowing rate (FR), ranging between 80.0% and 84.0%, and the total number of piglets born (TNB), ranging between 12.7 and 13.1, were recorded and from these the least square means per ejaculate were calculated. Only 5.9% of the total variation in FR was due to boar and semen variability of which 21% (P = 0.0001) was explained by genetic line of the boar, 11% (P = 0.047) was explained by laboratory technician, and 7% (P = 0.037) was explained by the AI center. For TNB the total variation was 6.6% boar and semen related of which 28% (P < 0.0001) was explained by genetic line of the boar and 7% (P = 0.011) was explained by the AI center. Only 4% of the boar and semen related variation was caused by sperm motility (microscopically assessed at collection, ranging from 60% to 90%). Other variation in FR and TNB was explained by management and semen related parameters (age of boar, 3%; P = 0.009; and 8%; P = 0.031, respectively), days between ejaculations (1%; P < 0.0001 of FR), number of cells in ejaculate (1%; P = 0.042 of TNB), year (9%; P = 0.032), and 13%; P = 0.0001, respectively), and month (11%; P = 0.0001; and 5%; P = 0.0001, respectively). Although semen motility is considered an important parameter to validate the quality of the ejaculate processed, it only minimally relates to fertility results under the current Dutch AI practice. Other boar and semen related parameters, like genetic line of the boar, are more relevant factors to select boars for AI purposes.

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells In-vitro - Corrected Proof

2012-01-30

Abstract: Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.

Vaginal bacterial flora and cytology in proestrous bitches: role on fertility - Corrected Proof

D. Groppetti, A. Pecile, C. Barbero, P.A. Martino — 2012-01-30

Abstract: The study of canine vaginal cytology underwent limited evolution over the years. Presence and significance of inflammatory cells in vaginal smears are little considered aspects in the bitch. Moreover, occurrence of vaginal bacteria in breeding bitches during follicular phase of the reproductive cycle, in absence of clinical signs of infection, involves the difficult question of antibiotics administration. The aim of this study was to relate findings in vaginal cytology (presence of neutrophils, lymphocytes, eosinophils, erytrocytes and bacteria) and microbial environment during proestrus with fertility outcomes (development of pregnancy, uterine infection, resorption, abortion and neonatal mortality). Bacteria sensitivity to antibiotics normally used in small animal practice was also evaluated. Bacteria isolated from vagina, in order of frequency, were Enterococcus faecalis, Streptococcus β-haemolyticus, Pasteurella multocida, E. coli, Klebsiella pneumoniae, Proteus mirabilis, E. coli haemolyticus, Arcanobacterium pyogenes, Streptococcus spp., Staphylococcus spp. and Acinetobacter spp. No mycoplasmas were observed. The present study showed that proestrous cytological aspects do not affect fertility. Eosinophils were never detected, while erythrocytes were always detected. During diestrus, E. coli was found in all pregnant bitches that developed clinical symptoms of uterine disorders (n = 3), resulting in uterine infection, resorption or abortion, but without statistical significance. Vaginal presence of Streptococcus spp. in proestrus was instead negatively associated with development of uterine infections (P = 0.005). Therefore, Streptococcus spp. could have a protective competitive role against more dangerous pathogens affecting fertility of the bitch. Among the 12 antibiotics tested, Gram-negative bacteria showed a significant sensitivity towards the amoxicillin and clavulanic acid association (P = 0.038). However, antibiotic treatment before mating, on the basis of positive culture, yet in the absence of clinical signs, seems to be unnecessary besides harmful leading to imbalance in vaginal commensal flora with adverse effects on fertility. In conclusion, vaginal bacteria, neutrophils, lymphocytes and erytrocytes should be considered as physiological aspect in the bitch during proestrus that does not require antibiotic therapy when asymptomatic.

Effect of antioxidants resveratrol and quercetin on in vitro evaluation of frozen ram sperm - Corrected Proof

E.C.B. Silva, J.F.P. Cajueiro, S.V. Silva, P.C. Soares, M.M.P. Guerra — 2012-01-30

Abstract: The objective was to assess the effects of the antioxidants resveratrol and quercetin on frozen-thawed ram sperm. Semen samples (which exceeded minimum standards) from four mature crossbreed Santa Inês rams were pooled and aliquots of each pool were diluted in Tris-egg yolk-glycerol, with the addition of 0, 5, 10, 15, and 20 μg/mL of resveratrol and quercetin in Experiment 1 and Experiment 2, respectively. In Experiment 1, the proportion of sperm with a high mitochondrial membrane potential was greater (P < 0.02) in the control group than in resveratrol 20 μg/mL group. In Experiment 2, the proportion of sperm with high mitochondrial membrane potential was greater in the control group (P < 0.0001) than in the other experimental groups, and greater in the quercetin 5 μg/mL group (P < 0.05) than in the other quercetin-treated groups. Thus, addition of 5 to 20 μg/mL of either resveratrol or quercetin to the Tris-egg yolk-glycerol extender reduced sperm mitochondrial membrane potential.

Effect of timing of hormonal induction on reproductive activity in lambari (Astyanax bimaculatus) - Corrected Proof

2012-01-30

Abstract: The objective was to evaluate the influence of the timing of hormonal induction, using gonadorelin or common carp pituitary extract (CPE), on the reproductive activity of female Astyanax bimaculatus. Fish (N = 44) were weighed, measured, and acclimatized to experimental conditions with a photoperiod of 12 h:12 h light:dark (L:D) for 10 days. Ovulation was induced with a single dose of CPE (6 mg/kg) or gonadorelin (80 μg/kg), given at 12:00 (halfway through the light phase (LP) or 24:00 (halfway through the dark phase (DP), in a 2 × 2 factorial design. The time of ovulation was calculated in degree hours and daily motor activity was recorded using a photocell. The fish were killed and the liver and gonads were weighed for calculation of gonadosomatic (GSI) and hepatosomatic (HSI) indexes, respectively. Absolute fecundity (AF), absolute fecundity relative to weight (AFRW) and length (AFRL), diameter of oocytes (mM), and percentage of oocytes with the germinal vesicle in a peripheral position (PPGV) were recorded. All females responded (ovulated). The female Astyanax bimaculatus had twilight motor activity rhythm. Females given CPE at 12:00 had a higher (P < 0.05) percentage of oocytes with the germinal vesicle in a peripheral position compared with the group that received gonadorelin in the same period (95 ± 6 vs. 79 ± 21%, mean ± SD). The absolute fecundity relative to weight was higher in groups induced at 12:00, regardless of the hormone used (LP: 805 ± 448 and 700 ± 214, for CPE and gonadorelin, respectively; dark phase: 580 ± 396 and 529 ± 105, P < 0.05). Both times used for hormonal induction with CPE and gonadorelin were suitable for inducing reproduction in lambari, although induction with CPE in LP had the best results.

Evaluation of ram semen quality using polyacrylamide gel instead of cervical mucus in the sperm penetration test - Corrected Proof

2012-01-30

Abstract: Fertility is a very complex biological function that depends on several properties of the spermatozoa, including sperm motility. Two objectives are analyzed in this study: (1) Replace the cervical mucus by a synthetic medium in a sperm penetration test, and (2) evaluating the results of this test objectively analyzing the sperm number that migrates. In experiment 1, we have tested eight concentrations of acrylamide (1%–2%). Rheological properties of media were analyzed. The plastic straws, loaded with acrylamide, were placed vertically on the semen sample tube for 15 min at 39 °C. After, the acrylamides were placed, by segments of 5 mm, into wells of a 24-well plate, dyed with Hoechst 33342 and the number of spermatozoa were calculated by automated microscopy analysis. The 1.55% and 1.6% acrylamide gel showed a number of spermatozoa emigrating closer to that seen with natural mucus. In experiment 2, we applied the sperm penetration in acrylamide 1.6% and 1.55% using fresh semen and cooled semen at 15 °C and 5 °C. The spermatozoa counts were performed for each segment of 10 mm. Semen chilled at 15 °C presented intermediate values of sperm counts in comparison with fresh semen (higher) and 5 °C chilled semen. The sperm counts do not differ between acrylamides but the rheological properties of acrylamide 1.6% were more similar to those of the natural cervical mucus. In experiment 3, we have observed significant correlations between the number of spermatozoa and several sperm quality parameters (positive: progressive motility and velocity according to the straight path; negative: damaged acrosomes and apoptotic cells) in 1.6% acrylamide media. We conclude that the size of the cell subpopulation, objectively calculated, that migrate beyond 20 mm in 0.5-mL straws filled with acrylamide is a useful parameter in ram sperm quality assessment and further studies are needed to evaluate its relationship with field fertility.

Erratum to “A rapid improved method for sexing embryo of water buffalo” Theriogenology 76 (2011) 83–87 - Corrected Proof

K.M.A. Zoheir, A.A. Allam — 2012-01-30

The above paper was published with an error in the Acknowledgements on page 87. The Acknowledgement should have read: “This project was supported by Research Centre, King Saud University, College of Pharmacy, Saudi Arabia, and National Research Centre, Cell Biology Department, Cairo, Egypt.” We regret any inconvenience that this error has caused.

Artificial insemination at 56 h after intravaginal progesterone device removal improved AI pregnancy rate in beef heifers synchronized with five-day CO-Synch + controlled internal drug release (CIDR) protocol - Corrected Proof

R. Kasimanickam, M. Asay, P. Firth, W.D. Whittier, J.B. Hall — 2012-01-30

Abstract: The objective was to determine whether timed artificial insemination (TAI) 56 h after removal of a Controlled Internal Drug Release (CIDR, 1.38 g of progesterone) insert would improve AI pregnancy rate in beef heifers compared to TAI 72 h after CIDR insert removal in a 5-days CO-Synch + CIDR protocol. Angus cross beef heifers (n = 1098) at nine locations [WA (5 locations; n = 634), ID (2 locations; n = 211), VA (one location; n = 193) and WY (one location; n = 60)] were included in this study. All heifers were given a body condition score (BCS; 1-emaciated; 9-obese), and received a CIDR insert and 100 μg of gonadorelin hydrochloride (GnRH) on Day 0. The CIDR insert was removed and two doses of 25 mg of dinoprost (PGF2α) were given, first dose at CIDR insert removal and second dose 6 h later, on Day 5. A subset of heifers (n = 629) received an estrus detector aid at CIDR removal. After CIDR removal, heifers were observed thrice daily for estrus and estrus detector aid status until they were inseminated. Within farm, heifers were randomly allocated to two groups and were inseminated either at 56 h (n = 554) or at 72 h (n = 544) after CIDR removal. All heifers were given 100 μg of GnRH at AI. Insemination 56 h after CIDR insert removal improved AI pregnancy rate compared to insemination 72 h (66.2 vs. 55.9%; P < 0.001; 1 – β = 0.94). Locations, BCS categories (≤ 6 vs. > 6) and location by treatment and BCS by treatment interactions did not influence AI pregnancy rate (P > 0.1). The AI pregnancy rates for heifers with BCS ≤ 6 and > 6 were 61.8 and 60.1%, respectively (P > 0.1). The AI pregnancy rates among locations varied from 54.9 to 69.2% (P > 0.1). The AI pregnancy rate for heifers observed in estrus at or before AI was not different compared to heifers not observed in estrus [(65.4% (302/462) vs. 52.7% (88/167); P > 0.05)]. In conclusion, heifers inseminated 56 h after CIDR insert removal in a 5-days CO-Synch + CIDR protocol had, on average, 10.3% higher AI pregnancy rate compared to heifers inseminated 72 h after CIDR insert removal.

Effects of resynchronization strategies for lactating Holstein cows on pattern of reinsemination, fertility, and economic outcome - Corrected Proof

2012-01-27

Abstract: The objectives were to evaluate the pattern of re-insemination, pregnancy outcomes to re-insemination in estrus and at fixed time, and economic outcomes of lactating Holstein cows submitted to three resynchronization protocols. Cows were enrolled in the Experiment at 32 ± 3 d after pre-enrollment Artificial Insemination (AI), 7 d before pregnancy diagnosis, and randomly assigned to three resynchronization protocols. All cows diagnosed not pregnant at 39 ± 3 d after pre-enrollment AI were submitted to the Cosynch72 (Day 0 GnRH, Day 7 prostaglandin F2α, and Day 10 GnRH and fixed time AI). Cows assigned to the control treatment received no further treatment, cows assigned to the GGPG treatment received a GnRH injection on Day −7, and cows assigned to the CIDR treatment received a controlled internal drug release (CIDR) insert containing 1.38 g of progesterone from Days 0–7. Cows observed in estrus were re-inseminated on the same day. Pregnancy was diagnosed at 39 ± 3 and 67 ± 3 d after re-insemination. Costs of the resynchronization protocols were calculated for individual cows enrolled in the study and pregnancies generated were given a value of $275. The GGPG treatment resulted in the slowest (P ≤ 0.06) rate of re-insemination. Overall pregnancy per AI (P/AI) at 39 ± 3 (P = 0.50) and 67 ± 3 (P = 0.49) d after re-insemination were not affected by treatment. Although cost of the control protocol was (P < 0.01) the smallest, return per cow resynchronized was (P < 0.01) greater for GGPG and CIDR protocols. We concluded that presynchronizing the estrous cycle of cows with GnRH or treating cows with a CIDR insert during resynchronization altered the pattern of re-insemination and improved the economic return of resynchronized cows.

Effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils (Meriones unguiculatus) - Corrected Proof

Xiaohui Lv, Y. Guo, D. Shi — 2012-01-27

Abstract: Quinestrol, a synthetic estrogen with marked estrogenic effects and prolonged activity, has potential as a contraceptive for Mongolian gerbils. The objective of this study was to describe the effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils. Serum and pituitary concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were decreased, whereas serum concentrations of estradiol (E2) and progesterone (P4) were increased after quinestrol treatment; the effects were both time- and dose-dependent. Furthermore, quinestrol downregulated expression of FSHβ and LHβ mRNA in the pituitary gland, as well as FSH receptor (FSHR) and estrogen receptor (ER) β in the ovary. However, it up-regulated mRNA expression levels of ERα and progesterone receptor (PR) in the pituitary gland and uterus, as well as mRNA for LH receptor (LHR) and PR in the ovary (these effects were time- and dose-dependent). In contrast, quinestrol had no significant effects on the mRNA expression levels of ERα in the ovary, or the gonadotropin α (GtHα) subunit in the pituitary gland. We inferred that quinestrol impaired synthesis and secretion of FSH and LH and that the predominant ER subtype in the pituitary gland of Mongolian gerbils may be ERα. Overall, quinestrol disrupted reproductive hormone receptor expression at the mRNA level in the pituitary-gonadal axis of the Mongolian gerbil.

Establishment of an efficient somatic cell nuclear transfer system for production of transgenic pigs - Corrected Proof

G. Vajta, H. Callesen — 2012-01-27

Abstract: Handmade cloning (HMC) is now an established procedure used in several species for somatic cell nuclear transfer, but only applied in two related laboratories for pigs. The aim of this review is to facilitate widespread application by summarizing the process of establishment and explaining the background of the incorporated special approaches. Optimized steps of traditional cloning in pigs (in vitro maturation, activation, embryo culture) were merged with those of the micromanipulation-free HMC that has been modified according to the specific needs of sensitive porcine oocytes (partial zona digestion before enucleation, two-step zona-free fusion with the somatic cell; initiation of activation with the second fusion). The zona-free approach required embryo culture to the blastocyst stage before surgical transfer of embryos to the uterine horns of recipient sows in the proper phase of an unstimulated cycle. Eventually a competitive, inexpensive and reliable alternative to traditional porcine nuclear transfer cloning techniques evolved that is also suitable to produce transgenic offspring containing various genetic modifications to establish models for several human diseases with genetic background. Further improvements and involvement of additional techniques to increase the overall efficiency and facilitate practical applications are expected in the foreseeable future.

Detection of the activator cAMP responsive element modulator (CREM) isoform ortholog proteins in porcine spermatids and sperm - Corrected Proof

T. Noda, O. Shidara, H. Harayama — 2012-01-27

Abstract: It is necessary to obtain basic information on transcription factors expressed in the spermatids of livestock to determine mechanisms of defective spermiogenesis. In this study, we characterized the activator cAMP responsive element modulator (CREM) ortholog isoforms in porcine testicular germ cells and ejaculated sperm. At least two kinds of porcine activator CREM τ family ortholog mRNAs were more strongly expressed in the testis than in kidney or liver. The activator CREM isoform ortholog proteins were localized in nuclei of round spermatids and around nuclei of elongated spermatids. Furthermore, approximately 34% of ejaculated sperm had the activator CREM isoform ortholog proteins at their connecting piece. This is apparently the first report demonstrating the expression and localization of the activator CREM isoform ortholog proteins in spermatids and ejaculated sperm of a livestock species.

Characterization of Na+K+-ATPase in bovine sperm - Corrected Proof

Katie D. Hickey, Mary M. Buhr — 2012-01-27

Abstract: Existing as a ubiquitous transmembrane protein, Na+K+-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na+K+-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na+K+-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na+K+-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na+K+-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm.

Osteo-regenerative potential of ovarian granulosa cells: An in vitro and in vivo study - Corrected Proof

2012-01-27

Abstract: Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.

Transrectal combined thickness of the uterus and placenta in normal pregnant Egyptian buffalo-cows - Corrected Proof

H. Zaher, H. Abdalla, F. Labib, A. Eidaroos — 2012-01-27

Abstract: The combined thickness of the uterus and placenta (CTUP) is one of the characteristics that can be used to assess fetal development and/or placental function in bovine. The current study was designed to establish reference values for the CTUP throughout pregnancy in normal pregnant buffalo-cows. The CTUP at the intracotyledonary space was measured monthly from the second month until full term using electronic calipers of the ultrasound machine. The CTUP increased monthly from 2.5 mm at the second month to 12 mm at the full term. During the last trimester, the monthly increase in the CTUP was higher than that recorded during the first and second trimesters. The result of the current study can be used as normal values for future studies of CTUP in pathologically pregnant buffalo-cows.

Role of LH in luteolysis and growth of the ovulatory follicle and estradiol regulation of LH secretion in heifers - Corrected Proof

O.J. Ginther, F.A. Khan, M.A. Hannan, M.B. Rodriguez, G. Pugliesi, M.A. Beg — 2012-01-27

Abstract: The role of LH in luteolysis and development of the ovulatory follicle and the involvement of GnRH receptors in estradiol (E2) stimulation of LH secretion were studied in heifers. A pulse of PGF2α, as indicated by a metabolite, was induced by E2 treatment on Day 15 (Day 0 = ovulation) and LH concentration was reduced with a GnRH-receptor antagonist (acyline) on Days 15, 16, and 17. Blood samples were collected every 6 h on Days 14–17 and hourly for 10 h beginning at the Day-15 treatments. Four groups were used (n = 6): control, acyline, E2, and E2/acyline. The number of LH pulses/heifer during the 10 h posttreatment was greater (P < 0.0002) in the E2 group (2.3 ± 0.4, mean ± SEM) than in the acyline group (0.2 ± 0.2) and was intermediate in the E2/acyline group (1.4 ± 0.2). Concentrations of progesterone in samples collected every 6 h on Day 15 showed a group-by-hour interaction (P < 0.02); concentrations decreased in the acyline group but not in the control group. The 12 heifers in the combined acyline and E2/acyline groups had three follicular waves compared to two waves in 10 of 12 heifers in the combined control and E2 groups. Results (1) supported the hypothesis that LH delays the progesterone decrease associated with luteolysis, (2) supported the hypothesis that LH has a positive effect on the continued development and growth of the selected ovulatory follicle, and (3) indicated that E2 stimulates LH production through an intracellular pathway that involves GnRH receptors on the gonadotropes and a pathway that does not involve the receptors.

Localization of tumor necrosis factor in the canine testis, epididymis and spermatozoa - Corrected Proof

R. Payan-Carreira, I. Santana, M.A. Pires, B. Ström Holst, H. Rodriguez-Martinez — 2012-01-12

Abstract: Tumor necrosis factor (TNF), formerly known as Tumor necrosis factor alpha is now regarded as a natural component of the mammalian seminal plasma (SP). Although not completely clarified, its functions in the SP have been associated with paradoxal roles, such as sperm survival in the female genital tract, while at high levels negatively affect sperm survival and fertility potential. Recently, it has been discovered that canine inseminated spermatozoa display a strong immunoreaction for TNF when lining the female endometrium. As a continuation of this finding, the present work aimed at documenting TNF localization in the canine testes and epididymis and in freshly ejaculated spermatozoa (SPZ) through immunohisto- or cytochemistry. Immunoreaction for TNF was found in all samples used. In the dog testis, TNF immunoexpression was limited to the seminiferous tubules, where late round spermatids (SPD) showed weak intensity of immunostaining, while elongating and elongated SPD evidenced moderate and the residual bodies a strong intensity. In the epididymis, a gradual progressive increase of TNF immunolabelling was found throughout the epididymal regions, ranging from a weak intensity at the caput epididymis to a moderate intensity at the cauda. TNF immunolabelling was found in mature SPZ during the epididymal transit and also in freshly ejaculated SPZ, which showed a strong midpiece immunolabelling. Data presented here provide important information on expression of TNF in spermatozoa, which is acquired by the SPZ during their formation at the testis. It further provides the basis for subsequent studies on the physiological importance of cytokines in sperm function.

Subpopulation distribution of motile sperm relative to activation medium in steelhead (Oncorhynchus mykiss) - Corrected Proof

M.K. Kanuga, R.E. Drew, J.G. Wilson-Leedy, R.L. Ingermann — 2012-01-09

Abstract: In the present study, steelhead sperm were activated in artificial tap water, ovarian fluid, activating saline, or in combinations of these media, and motility characteristics were determined using computer-assisted sperm analysis. Motility characteristics of individual sperm were then assessed to test the hypothesis that motile sperm are distributed among discrete subpopulations and that their distribution is influenced by the activation medium. Analysis with k-means clustering detected three discrete motile sperm subpopulations in steelhead semen, regardless of the activation medium. Based on multivariate analysis of variance, proportions of these subpopulations did not differ between sperm activated with ovarian fluid and activating saline, or any combination of these two media. However, subpopulation distributions for sperm activated with either ovarian fluid or activating saline were influenced by the level of dilution of these media in artificial tap water. There was an increase in the number of sperm in high velocity (curvilinear), high straightness, and high wobble subpopulation with increased levels of ovarian fluid or activating saline. The change in sperm motility characteristics with a change in activation medium may play a role in normal fertilization, as discharged sperm pass from seminal plasma and water through ovarian fluid en route to the egg.

The dynamics of sperm DNA stability in Asian elephant (Elephas maximus) spermatozoa before and after cryopreservation - Corrected Proof

2012-01-09

Abstract: The purpose of this study was to investigate the occurrence of sperm DNA fragmentation in Asian elephant (Elephas maximus) spermatozoa at various processing stages before and after cryopreservation. Five semen samples from four elephants were assessed at four different stages during processing; after (1) collection and reextension in TEST-egg yolk; (2) cooling to 5 °C; (3) equilibration for 1 h with glycerol; (4) thawing. An experimental approach was adopted that allowed comparisons of DNA fragmentation rates developed after the various processing stages. For this, spermatozoa were incubated in TEST-yolk media at 37 °C for 0, 4, 8, 24 and 48 h, and sperm DNA fragmentation rates were estimated using an elephant-specific Halosperm procedure. Incubation at 37 °C induced a rapid increase in DNA fragmentation, and significant differences between males were observed. The overall rate of increase over 4 h was estimated at about 5% per hour, and no significant changes to this rate were observed at the different processing stages, even, including the post-thaw samples. As semen quality of the five ejaculates was relatively poor, the basic semen parameter data were compared with nine different samples collected 11 mo earlier to see whether the tested samples were atypical or representative of the population, As there was no significant difference between the two sets of samples, it is believed that the samples tested for DNA stability were not unusually sensitive. These results suggest that Asian elephant spermatozoa are more susceptible to DNA fragmentation than spermatozoa of other mammals.

Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation - Corrected Proof

2012-01-09

Abstract: The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes.

Cervical expression of hyaluronan synthases varies with the stage of the estrous cycle in the ewe - Corrected Proof

K. Perry, W. Haresign, D.C. Wathes, A.A. Pitsillides, M. Khalid — 2012-01-09

Abstract: The natural cervical relaxation which occurs at estrus in the ewe may be initiated by binding of hyaluronan (HA) to its receptor CD44. Indeed, we have previously shown that HA content and fragment size in the ovine cervix varies with the stage of the estrous cycle. Despite the importance of cervical relaxation in promoting sperm transport and facilitating the possible development of transcervical artificial insemination (AI), the mechanisms coordinating these changes in HA content remain to be defined. Hyaluronan synthases (HAS) 1, 2, and 3 regulate HA biosynthesis and herein, we describe the changing pattern of HAS isoform expression during the estrous cycle to determine whether this may underpin HA-mediated changes in relaxation of the ovine cervix. Accordingly, cervices were collected from 24 cyclic sheep (n = 8 / group) at the luteal, pre-luteinizing hormone (LH) and post-LH surge stages. Protein and mRNA expression for HAS 1, 2 and 3 was determined in five different tissue layers (epithelium, subepithelial stroma, and longitudinal, circular and transverse muscle) of the vaginal, mid and uterine regions of each cervix by immunohistochemistry and in situ hybridization, respectively. HA synthases were expressed in all the tissue layers and regions of the cervix, and the pattern of expression was similar for mRNA and protein. HAS1 protein and mRNA expression was significantly (P ≤ 0.05) higher at the pre-LH surge stage, while HAS 2 and 3 protein and mRNA expression was significantly (P ≤ 0.001) higher at the luteal stage. Overall, both HAS protein and mRNA expression was significantly (P ≤ 0.001) higher in the epithelial layer and the vaginal region. These findings are in accordance with our previous results and explain the differences observed in the HA content and differing HA fragment size at different stages of the estrous cycle.

The effects of bovine necrotic vulvo-vaginitis on reproductive and production performance of Israeli 1st calf heifers - Corrected Proof

T. Goshen, J. Ben-Gera, O. Koren, T. Bdolah-Abram, D. Elad — 2012-01-09

Abstract: Bovine necrotic vulvovaginitis (BNVV) is a syndrome unique to Israel characterized by necrotic lesion in the caudal vagina mainly in first calf heifers after calving, associated with Porphyromonas levii. The objectives of this study were to analyze the impact of BNVV on reproductive performance, milk production and survival in the heard of first calf dairy heifers in affected farms, and to verify if the effects of BNVV are severity-dependent. For assessment of the severity level a scale of 4 degrees was formed, and cows were scored 4 to 6 d after calving. Data were obtained from two dairy farms during 2006–07, consisting of 603 lactations. The incidence and the severity of BNVV declined between 2006 and 2007, and severe BNVV tended to be more prevalent in the summer. The odds to conceive in the first artificial insemination of BNVV cow tended to be lower than healthy cows (OR = 0.676, P = 0.052). Cows with BNVV had longer empty period (145.8 d vs. 135.1 d of healthy cows, P = 0.031), but only severe BNVV had a negative effect on the odds of the cow to be empty at 150 d in milk (DIM) (OR = 2.05, P = 0.052). Severe BNVV also affected the mean survival time to conception (155.9 d vs. 142.3 d, P = 0.042). All BNVV severity degrees had a negative effect on milk production. The effect on milk production was not limited only to the beginning of the lactation, cows with BNVV produced 338.1 kg milk less than healthy cows (P = 0.016) in 305 d corrected lactation. The effect on milk production was not severity depended. No effect on survival time in the herd was demonstrated.

The influence of 9-cis-retinoic acid on nuclear and cytoplasmic maturation and gene expression in canine oocytes during in vitro maturation - Corrected Proof

2012-01-09

Abstract: Retinoids have important roles in regulation of oocyte nuclear and cytoplasmic maturation. The present study investigated the effects of a retinoid metabolite on nuclear maturation, cytoplasmic maturation, and gene expression in canine oocytes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were harvested from ovaries by slicing. Only oocytes that were >120 μm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. Varying concentrations of 9-cis retinoic acid (9-cis-RA; 0, 5, 50, and 500 nm) were included in the maturation medium, and the following were measured: (i) oocyte nuclear maturation after culture for 48 h; (ii) cytoplasmic granular migration by labeling of oocytes with fluorescein isothiocyanate labeled lectins; and (iii) relative expression of genes related to apoptosis (BAX and BclII) in cumulus cells detached from oocytes, by semiquantitative reverse transcriptase-polymerase chain reaction. After 48 h culture with IVM, the highest percentage of oocytes that had developed to the metaphase II (MII) stage were in the 5 nm 9-cis-RA treatment group (18.3 ± 2.5%; P < 0.05). Complete granular migration was observed in oocytes matured with 5 nm 9-cis-RA, consistent with a commensurate gain in developmental competence. Treatment with 5 nm 9-cis-RA had no effect on BclII gene expression, but downregulated BAX expression. In conclusion, since 5 nm 9-cis-RA was beneficial to nuclear and cytoplasmic maturation of canine oocytes, we inferred an important role for 9-cis-RA during IVM.

Influence of day of postpartum breeding on pregnancy rate, pregnancy loss rate, and foaling rate in Thoroughbred mares - Corrected Proof

2012-01-09

Abstract: Records (years 2005–2007) were analyzed from a Thoroughbred stud farm in central Kentucky. Data from all breeding cycles of foaling mares were tabulated (3184 cycles of 2003 foaling mares bred between 7 and 163 days postpartum). A multiple logistic regression model employing Bayesian statistics was used to adjust for factors that significantly affected outcome; odds ratios (ORs) for pregnancy rate, pregnancy loss rate, and foaling rate were determined to examine the influence of day of postpartum breeding on these parameters. Mares bred before Day 22 (Day 0 = day of foaling) postpartum had a decreased OR for becoming pregnant (P < 0.05); the median OR for becoming pregnant (1.00) was not reached until Day 46 postpartum. Mares bred before Day 13 postpartum had an increased OR for pregnancy loss (P < 0.05). The median OR for pregnancy loss did not decline below 1.00 until Day 78 postpartum. Mares bred before Day 20 postpartum had a decreased OR for producing a foal (P < 0.05). The median OR for producing a foal (1.00) was not reached until Day 75 postpartum. We concluded that fertility (in terms of a higher OR for becoming pregnant and a lower OR for pregnancy loss, resulting in a higher OR for producing a foal) continued to improve in Thoroughbred mares for approximately 2.5 mo postpartum. These findings are of importance to management strategies directed at early postpartum breeding, and explain some of the reported drift in subsequent foaling dates of Thoroughbred mares, despite management practices that employ early postpartum breeding.

Variations of chromatin, tubulin and actin structures in primate oocytes arrested during in vitro maturation and fertilization—what is this telling us about the relationships between cytoskeletal and chromatin meiotic defects? - Corrected Proof

S. Delimitreva, O.Y. Tkachenko, A. Berenson, P.L. Nayudu — 2012-01-09

Abstract: A nonhuman primate model was applied to investigate the relationships between variations in the organization of microtubules, microfilaments, and chromatin in metaphase I and metaphase II oocytes. Marmoset oocytes were subjected to in vitro maturation and coincubation with sperm. Oocytes which failed to cleave were investigated for chromatin, tubulin, and actin using Hoechst 33258, fluorescein isothiocyanate (FITC)-labeled alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. Spindles were categorized according to size, shape and microtubule organization: normal, large, multipolar, disorganized, absent spindle, and spindles with broad poles. Actin caps were categorized as: normal, small, split, and disorganized. Chromosomal condensation and alignment were described as normal or abnormal. Improper chromosomal condensation was associated with both abnormal microfilament and microtubule arrangement. This was further associated with abnormal actin organization, disorientation and late stabilization of microtubules, but not related to abnormal organization of spindle poles. Chromosomal misalignment was associated with disorientation and late stabilization of tubulin, but not to broad spindle pole. Additionally, abnormal actin polarization appeared not to be related to abnormal spindle poles. The model system presented in this study could be used as an experimental platform for studying the contribution of different factors to the exactness of late meiotic events in primate oocytes. The present study provides basic information on spindle, chromosome, and actin normal and abnormal organization, which can be observed in in vitro matured, but failed to cleave primate oocytes.

Nitric oxide in the bovine oviduct: Influence on contractile activity and nitric oxide synthase isoforms localization - Corrected Proof

2012-01-09

Abstract: The oviducts of 64 Holstein cows in luteal (early I, early II and late) and follicular phases were evaluated to determine the protein expression and mRNA transcription of different nitric oxide synthase isoforms (eNOS, iNOS, nNOS) as well as the effect of nitric oxide (NO) on spontaneous contractility in vitro. The expression patterns of nitric oxide synthase (NOS) isoforms in isthmus and ampulla (n = 6 for each phase) were determined by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. In the contractility studies, longitudinal and circular isolated strips of isthmus and ampulla (n = 10 for each phase) of oviducts located ipsilateral to the luteal structure or preovulatory follicle were treated as follows: a) L-arginine, an endogenous NO donor (10−8 to 10−3 m), b) Nω-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (10−5 m) and L-arginine (10−3 m), c) methylene blue (MB), an inhibitor of soluble guanylate (10−5 m) and L-arginine (10−3 m) and d) sodium nitroprusside (SNP), an exogenous NO donor (10−8 to 10−4 m). Immunohistochemical evaluation revealed that endothelial NOS (eNOS) expression detected in epithelial layer of isthmus and ampulla was strong in early I luteal phase, moderate in follicular phase and weak in other phases. Neuronal NOS (nNOS) immunoreactivity was strong in isthmus and moderate in ampulla, and staining of nerve fibers was observed mostly in early I luteal and follicular phases. All eNOS, nNOS and inducible NOS (iNOS) isoforms were detected by RT-PCR. eNOS and iNOS proteins were evident, whereas nNOS was undetectable by Western blot analysis in the tissue examined. L-arginine applied alone or after L-NAME did not alter or increase the contractile tension of the strips in most tissues examined. However, L-arginine applied after MB increased contractile tension in the strips of ampulla and longitudinal isthmus from early I luteal phase and circular isthmus from follicular phase but decreased it in isthmus from early II luteal phase. SNP differentially modulated oviductal contraction depending on the type of muscular strips and period examined. These results showed the estrous phase-dependent changes related to endogenous NO system which might be of physiological importance to the oviduct for secretory and ciliary functions involved in gametes and embryo(s) transportation.

The influence of genital tract status in postpartum period on the subsequent reproductive performance in high producing dairy cows - Corrected Proof

I. López-Helguera, F. López-Gatius, I. Garcia-Ispierto — 2012-01-09

Abstract: The aim of the present study was to characterize the early postpartum period in clinically healthy dairy cows by ultrasonography (US), endometrial cytology (EC), and white blood cell counts, and determine possible relationships between postpartum findings and subsequent reproductive performance. Fifty-three dairy cows were examined on Days 15 to 21 (Visit 1), 22 to 28 (Visit 2), and 29 to 35 (Visit 3) postpartum. The clinical examination included: examination of vaginal fluid, EC, transrectal palpation and ultrasonography of the genital tract (cervical diameter, endometrial thickness, presence of a corpus luteum [CL] or intrauterine fluid [IUF] and its echogenicity). Luteal activity (presence of a CL in a single visit), return to cyclicity (presence of a CL in 2 consecutive visits), and conception rate at 70 and 120 days postpartum were considered as the dependent variables in four consecutive binary logistic regression analyses. Factors affecting leukocyte counts were established by general linear model (GLM) repeated measures analysis of variance. Based on the odds ratio (OR), the likelihood of luteal activity was higher in multiparous than primiparous cows (OR = 3.75) and tended to diminish in cows showing increased endometrial thickness in Visit 1 (V1) (OR = 0.06). The likelihood of returning to cyclicity decreased for each centimeter increase in cervical diameter in V1 (OR = 0.14) and that of conception on Day 70 was lower in cows showing the presence of echogenic or anechogenic IUF in V1 (OR = 0.09 or OR = 0.13, respectively) compared with cows lacking IUF. Effects of parity and IUF were observed on neutrophil counts. Positive EC results were unrelated to the cumulative conception rate at 70 and 120 days in milk, whereas cows returning a positive EC result in V1 showed a greater likelihood of increased endometrial thickness. In conclusion, measuring cervical diameter, endometrial thickness, and detecting the echogenicity of IUF by ultrasonography from Days 15 to 21 postpartum in clinically normal cows is an appropriate tool to predict subsequent reproductive performance. Vaginal examination and transrectal palpation alone did not emerge as valuable predictors.

Automatic evaluation of ram sperm morphometry - Corrected Proof

J.L. Yániz, S. Vicente-Fiel, S. Capistrós, I. Palacín, P. Santolaria — 2012-01-09

Abstract: This study was designed to develop a new method based on fluorescence microscopy and image analysis for the automatic assessment of sperm morphometry and to study separately the effect of drying and fixation on the parameters of head sperm morphometry in the ram. The study was divided into two experiments. In the first experiment, ejaculates from 25 adult males were collected using an artificial vagina, diluted and divided into four sample aliquots. The first was labeled directly with Hoechst 33342 (FRESH), and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying (DRIED), and the other were fixed either with glutaraldehyde (GLUT), or with methanol (MET), and labeled with Hoechst afterward. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and sperm heads were automatically captured and analyzed using the ImageJ program. The method used allowed a fast and automatic selection of most sperm heads for a given image with high precision. There was a general trend toward significant decrease in head length, width, area and perimeter of air-dried sperm compared with fresh sperm. On average, this decrease was of 4.1% in length, 4.3% in width, 9.1% in area, and 2.8% in perimeter. Between semen smears, fixation with glutaraldehyde significantly increased head sperm dimensions. The smears fixed with glutaraldehyde method is recommended for a more practical use than with fresh samples, providing better quality images than the other methods, and because the morphometric results obtained were more similar to the FRESH group than those of the DRIED and MET. In the second experiment, ejaculates from adult males were used to compare the sperm head morphometric results obtained with the new method developed (using the GLUT treatment as reference) with a more conventional CASMA method (semen smears stained with Hemacolor and processed with the ISAS commercial software, HEM). The GLUT method allowed the analysis of 100% of sperm, whereas only 93% of sperm could be analyzed using HEM. Spermatozoa displayed a bigger size when processed with HEM than with GLUT method in all primary sperm head morphometric parameters. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters. The new method developed allows automatic determination of sperm head morphometry in a reduced time, which facilitates its use in routine semen analysis. It was concluded that the automation of sperm morphometry is feasible using fluorescence microscopy and image analysis and that the effect of drying and fixation was less important than previously stated.

Boar sperm thawing practices: the number of straws does matter - Corrected Proof

I. Casas, E. Torner, M. Yeste, S. Bonet — 2012-01-09

Abstract: The number of straws thawed has been largely neglected in reports of boar sperm cryopreservation. Whereas previous studies confirm the effect of sperm concentration on function and survival of thawed boar spermatozoa, it is still unknown whether, for a same concentration, total number of sperm in the thawing solution affects its mechanics. The present trial sought to define good boar sperm thawing practices by checking if a minimal number of straws as well as the percentage of air volume in the thawing tube should be stated or not to decrease variability from one trial to another. In a first assay, three tubes with different numbers of thawed straws were compared in terms of motility and membrane integrity: control (C, four straws), T1.1 (two straws), and T1.2 (one straw). In a second parallel assay, the sperm motility was evaluated when one straw was thawed in a tube containing 86.67% of air volume (T2.1), and when the tube contained < 1% air volume (T2.2). In all treatments the final concentration of sperm in Beltsville thawing solution (BTS) was 1:3 (v:v) and quality parameters were assessed 4 h after thawing. Results showed the number of straws does affect motility parameters but not the membrane integrity, whereas less air volume in the tube nonsignificantly minimizes data deviation among replicates. In conclusion, it is recommended the use of four straws at 1:3 (v:v) to maintain motility records in boar sperm thawing practices as well as to be provided with vials that fit the sperm volume.

Effect of corticotherapy on proteomics of endometrial fluid from mares susceptible to persistent postbreeding endometritis - Corrected Proof

C.A. Wolf, E. Maslchitzky, R.M. Gregory, M.I.M. Jobim, R.C. Mattos — 2012-01-09

Abstract: The objective was to determine the effects of corticotherapy, in the presence and absence of uterine inflammation, on proteomics of endometrial fluid from mares susceptible to endometritis. In 11 mares, estrus was induced seven times with 5 mg PGF2α given at 14-day intervals. The first estrus was a control (no treatment). During the third estrus, mares received glucocorticoid (GC) treatment (20 mg isoflupredone acetate) every 12 h, for three consecutive days. The fifth estrus was the Infected treatment (intrauterine infusion of 1 × 109 colony-forming unit/mL Streptococcus equi subspecies zooepidemicus). Finally, the seventh was a combination of GC + Infected treatment (infusion of bacteria 24 h after the first GC treatment). At 12 h after the end of each treatment, uterine samples were collected and submitted to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and mass spectrometry. Both GC treatment and uterine lumen infection induced proteomic alterations in the endometrial fluid of susceptible mares, characterized by an increase, decrease, or both in the relative optic density and/or frequency of inflammatory acute phase proteins (APP), with major alterations occurring when corticotherapy was applied in the presence of an infectious process. Corticotherapy in the presence of infection increased α1-antitrypsin (AAT), transthyretin (TT), and actin, but reduced immunoglobulin G, whereas intrauterine infection increased haptoglobin (Hp) and apolipoprotein A-1 (ApoA-1) and decreased transferrin (TF). Infection reduced levels of α1-antitrypsin and transthyretin, whereas corticotherapy in the presence of infection increased their frequency. We concluded that GC influenced the immune response, not only as suppressors, but also as enhancers of local defense mechanisms, through an immunomodulatory action. Short-term corticotherapy could be beneficial for treatment of uterine infectious processes in the mare.

Comparison of heat transfer in liquid and slush nitrogen by numerical simulation of cooling rates for French straws used for sperm cryopreservation - Corrected Proof

M. Sansinena, M.V. Santos, N. Zaritzky, J. Chirife — 2012-01-09

Abstract: Slush nitrogen (SN2) is a mixture of solid nitrogen and liquid nitrogen, with an average temperature of −207 °C. To investigate whether plunging a French plastic straw (commonly used for sperm cryopreservation) in SN2 substantially increases cooling rates with respect to liquid nitrogen (LN2), a numerical simulation of the heat conduction equation with convective boundary condition was used to predict cooling rates. Calculations performed using heat transfer coefficients in the range of film boiling confirmed the main benefit of plunging a straw in slush over LN2 did not arise from their temperature difference (−207 vs. −196 °C), but rather from an increase in the external heat transfer coefficient. Numerical simulations using high heat transfer (h) coefficients (assumed to prevail in SN2) suggested that plunging in SN2 would increase cooling rates of French straw. This increase of cooling rates was attributed to a less or null film boiling responsible for low heat transfer coefficients in liquid nitrogen when the straw is placed in the solid–liquid mixture or slush. In addition, predicted cooling rates of French straws in SN2 tended to level-off for high h values, suggesting heat transfer was dictated by heat conduction within the liquid filled plastic straw.

Changes in sperm parameters of sex-reversed female rainbow trout during spawning season in relation to sperm parameters of normal males - Corrected Proof

2012-01-09

Abstract: The production of all-female populations has important economic benefits in commercial rainbow trout aquaculture. The procedure commonly implemented to produce all-female stocks centers on the sex reversal of rainbow trout females via the administration of androgens in the early developmental stages, followed by the egg fertilization of normal females with semen from sex-reversed females (srf). However, there is no information regarding the quality of semen from srf rainbow trout throughout the spawning season. This information is critical because the quality of srf semen is highly variable. The aim of the study was to determine the changes in the semen parameters of srf rainbow trout throughout the duration of the spawning season. Sperm concentration, sperm motility parameters, and the biochemical parameters of seminal plasma (protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity) from srf were monitored during the spawning season and compared with normal male rainbow trout. The observed values of sperm, protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity of seminal plasma were all higher in comparison with normal males. Semen from srf was therefore characterized by a lower sperm motility during each period of the spawning season, in comparison with normal males, approximately 1.8, 1.5, and 1.7 times, respectively for the beginning, middle, and end of the spawning season. The percentage of sperm motility from srf and normal males were affected by the spawning season in the same way, as the highest values in the middle of the spawning season demonstrate (60% and 91% for srf and normal males, respectively). Spermatozoa of srf are characterized by a lower speed and a more curvilinear trajectory of movement as compared with that of normal males. The patterns of changes during the spawning season in sperm concentration, sperm motility parameters, as well as osmolality, and lactate dehydrogenase activity of the seminal plasma of srf were different in comparison with normal males. Our results could be important for fish breeders in regard to the spawning control of srf rainbow trout, as well as for the development of short- and long-term sperm storage procedures.

The inseminating bull and plasma pregnancy-associated glycoprotein (PAG) levels were related to peripheral leukocyte counts during the late pregnancy/early postpartum period in high-producing Dairy cows - Corrected Proof

2012-01-09

Abstract: It has been established that the immunologic and endocrine status of the peripartum dairy cow determines the animal's subsequent productive and reproductive performance. Thus, at parturition reduced immune functions of peripheral blood polymorphonuclear cells (PMN) has been observed after a peak in pregnancy-associated glycoproteins (PAGs), and, more recently, the inseminating bull was linked to plasma levels of bovine PAGs in pregnant Holstein-Friesian dairy cows. The present study sought to determine whether changes in leukocyte counts during the peripartum period, indicative of the animal's immune status, could be related to the inseminating bull and to PAG levels. Ninety-six clinically healthy, single pregnant cows in a commercial dairy herd were selected. Four samples were collected before parturition (on gestation Days 220–226, 234–240, 248–254, and 262–268) and two samples after parturition (on Days 14–21, and 28–34 postpartum) to analyze total and differential blood cell counts. Based on GLM analysis procedures of variance for repeated measures, the inseminating bull was found to affect counts of total leukocytes and lymphocytes (P < 0.001; between-subject effects) throughout the peripartum period. In addition, cows with high plasma PAG levels (> 900 ng/ml) on Day 262–268 of gestation had higher numbers of total leukocytes and neutrophils throughout the peripartum (P < 0.001; between-subject effects). Young animals (≤ 1 lactation) had higher total leukocyte and lymphocyte counts than older cows (2 or more lactations) throughout the study period. These results reveal a clear relationship between the inseminating bull or plasma PAG levels and peripheral leukocyte counts during the peripartum period in dairy cows.

Effect of growth rate from 6 to 16 months of age on sexual development and reproductive function in beef bulls - Corrected Proof

L.F.C. Brito, A.D. Barth, R.E. Wilde, J.P. Kastelic — 2012-01-09

Abstract: Sexual development and reproductive function were studied in 22 Angus × Charolais and 17 Angus bulls from 6 to 16 mo of age. Associations of average daily gain (ADG) and body weight with ages at puberty and at maturity (satisfactory semen quality), scrotal circumference, paired-testes volume and weight, testicular vascular cone diameter and fat thickness, scrotal temperature, sperm production and morphology, and testicular histology, were determined. There were no significant correlations between cumulative average daily gain and any of the end points investigated. Body weight at various ages was negatively correlated with ages at puberty and maturity in Angus × Charolais bulls, positively correlated with paired-testes weight in Angus × Charolais and Angus bulls, and positively correlated with seminiferous tubule volume in Angus bulls (P < 0.05). Semen quality improved gradually with age and the interval between puberty and maturity (mean ± SD; 309.4 ± 29.7 and 357 ± 42 days of age) was approximately 50 days. Age, weight, scrotal circumference, and paired-testes volume were all good predictors of pubertal and mature status, with moderate to high sensitivity and specificity (71.6% to 92.4%). In summary, growth rate between 6 and 16 mo of age did not affect sexual development and reproductive function in beef bulls. However, greater body weight at various ages was associated with reduced age at puberty and maturity, and with larger testes at 16 mo of age, indicating that improved nutrition might be beneficial, but only when offered before 6 mo of age. Average daily gains of approximately 1 to 1.6 kg/day did not result in excessive fat accumulation in the scrotum, increased scrotal temperature, or reduction in sperm production and semen quality, and could be considered “safe” targets for growing beef bulls.

Effect of cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activity in fowl semen - Corrected Proof

Agnieszka Partyka, Ewa Łukaszewicz, Wojciech Niżański — 2012-01-09

Abstract: The aim of the present study was to determine the influence of chicken semen cryopreservation on sperm parameters, lipid peroxidation and antioxidant enzymes activities. Pooled semen from 10 Black Minorca roosters was used in the study. Semen samples were subjected to cryopreservation using the “pellet” method and dimethylacetamide (DMA) as a cryoprotectant. In the fresh and the frozen-thawed semen sperm membrane integrity (SYBR-14/propidium iodide (PI)), acrosomal damage (PNA-Alexa Fluor®488) and mitochondrial activity (Rhodamine 123) were assessed using flow cytometry. Malondialdehyde (MDA) concentration, catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were determined in sperm cells and seminal plasma by spectrophotometry. All sperm characteristics evaluated using flow cytometry were affected by cryopreservation. After freezing-thawing, there was significant (P < 0.01) reduction in sperm membrane integrity, sperm acrosome integrity and mitochondrial activity. Following cryopreservation, MDA concentration significantly increased in chicken seminal plasma and spermatozoa (P < 0.01, P < 0.05). The CAT activity in seminal plasma significantly decreased (P < 0.05), while intracellular activity of this enzyme did not significantly change in frozen-thawed semen. In seminal plasma of frozen-thawed semen the significant increase (P < 0.01) in GPx activity was detected. Whereas GPx activity in spermatozoa remained statistically unchanged after thawing. The SOD activity significantly increased (P < 0.01) in cryopreserved seminal plasma with simultaneous decrease (P < 0.01) of its activity in cells. In conclusion, this is probably the first report describing the level of antioxidant enzymes in frozen-thawed avian semen. The present study showed that the activity of CAT, GPx and SOD in chicken semen was affected by cryopreservation, what increased the intensity of lipid peroxidation (LPO). Catalase appeared to play an important role in the sperm antioxidant defense strategy at cryopreservation since, opposite to SOD and GPx, its content was clearly reduced by the cryopreservation process. Change in the antioxidant defense status of the chicken spermatozoa and surrounding seminal plasma might affect the semen quality and sperm fertilizing ability.

Lacking expression of paternally-expressed gene confirms the failure of syngamy after intracytoplasmic sperm injection in swamp buffalo (Bubalus bubalis) - Corrected Proof

2012-01-09

Abstract: The objectives were to: (1) examine the efficiency of intracytoplasmic sperm injection (ICSI) technique, with or without chemical activation of in vitro matured buffalo oocytes, on sperm head decondensation; and (2) compare the subsequent development of embryos after activation of ICSI (ICSI (+) activation group) and sham injection (Sham (+) activation group) oocytes (embryos obtained by in vitro fertilization of IVM oocytes served as a control group). Pronuclear formation rates in ICSI (+) activation and Sham (+) activation groups were higher than that of ICSI without activation (P < 0.05). However, because 90.9% of presumptive zygotes in ICSI (+) activation group demonstrated pronuclear formation with an intact sperm head, we inferred that most were parthenotes. Neither developmental competence (morula and blastocyst formation rates) nor mean total cell number of blastocysts was significantly different among ICSI (+) activation, Sham (+) activation, and IVF groups. To clarify whether blastocysts were derived from syngamy or parthenogenesis, expression of Nnat, a paternally expressed gene in blastocysts derived from IVF, ICSI and oocyte activation without sperm or sham injection was additionally examined using reverse transcription polymerase chain reaction (RT-PCR). Expression of Nnat mRNA was not detected in ICSI (+) activation blastocysts, indicating failure of male genome activation. Although blastocyst development after ICSI combined with chemical activation was similar to IVF oocytes, these blastocysts were generated by parthenogenesis, due to failure of male pronucleus formation.

Exploring the application of high-throughput genomics technologies in the field of maternal-embryo communication - Corrected Proof

Carmen Almiñana, Alireza Fazeli — 2012-01-05

Abstract: Deciphering the complex molecular dialogue between the maternal tract and embryo is crucial to increasing our understanding of pregnancy failure, infertility problems and in the modulation of embryo development, which has consequences through adulthood. High-throughput genomic technologies have been applied to look for a holistic view of the molecular interactions occurring during this dialogue. Among these technologies, microarrays have been widely used, being one of the most popular tools in maternal-embryo communication. Today, next generation sequencing technologies are dwarfing the capabilities of microarrays. The application of these new technologies has broadened to almost all areas of genomics research, because of their massive sequencing capacity. We review the current status of high-throughput genomic technologies and their application to maternal-embryo communication research. We also survey next generation technologies and their huge potential in many research areas. Given the diversity of unanswered questions in the field of maternal-embryo communication and the wide range of possibilities that these technologies offer, here we discuss future perspectives on the use of these technologies to enhance maternal-embryo research.

Parthenogenesis in non-rodent species: developmental competence and differentiation plasticity - Corrected Proof

T.A.L. Brevini, G. Pennarossa, A. Vanelli, S. Maffei, F. Gandolfi — 2012-01-05

Abstract: An oocyte can activate its developmental process without the intervention of the male counterpart. This form of reproduction, known as parthenogenesis, occurs spontaneously in a variety of lower organisms, but not in mammals. However, it must be noted that mammalian oocytes can be activated in vitro, mimicking the intracellular calcium wave induced by the spermatozoon at fertilization, which triggers cleavage divisions and embryonic development. The resultant parthenotes are not capable of developing to term and arrest their growth at different stages, depending on the species. It is believed that this arrest is due to genomic imprinting, which causes the repression of genes normally expressed by the paternal allele. Human parthenogenetic embryos have recently been proposed as an alternative, less controversial source of embryonic stem cell lines, based on their inherent inability to form a new individual. However many aspects related to the biology of parthenogenetic embryos and parthenogenetically derived cell lines still need to be elucidated. Limited information is available in particular on the consequences of the lack of centrioles and on the parthenote's ability to assemble a new embryonic centrosome in the absence of the sperm centriole. Indeed, in lower species, successful parthenogenesis largely depends upon the oocyte's ability to regenerate complete and functional centrosomes in the absence of the material supplied by a male gamete, while the control of this event appears to be less stringent in mammalian cells. In an attempt to better elucidate some of these aspects, parthenogenetic cell lines, recently derived in our laboratory, have been characterized for their pluripotency. In vitro and in vivo differentiation plasticity have been assessed, demonstrating the ability of these cells to differentiate into cell types derived from the three germ layers. These results confirmed common features between uni- and bi-parental embryonic stem cells. However data obtained with parthenogenetic cells indicate the presence of an intrinsic deregulation of the mechanisms controlling proliferation vs. differentiation and suggest their uni-parental origin as a possible cause.

Using computational modeling to investigate sperm navigation and behavior in the female reproductive tract - Corrected Proof

M. Burkitt, D. Walker, D.M. Romano, A. Fazeli — 2012-01-05

Abstract: The processes by which individual sperm cells navigate the length and complexity of the female reproductive tract and then reach and fertilize the oocyte is fascinating. Numerous complex processes potentially influence the transport of spermatozoa within the tract, resulting in a regulated supply of spermatozoa to the oocytes at the site of fertilization. Despite significant differences between species, breeds, and individuals, these processes converge to ensure that a sufficient number of high quality spermatozoa reach the oocytes, resulting in successful fertilization without a significant risk of polyspermy. Different factors, such as the physical complexity of the oviductal environment, changing swimming patterns, capacitation, chemotactic and thermotactic attraction, attachment and detachment from the oviductal epithelium, interactions with local oviductal secretions, individual variations in spermatozoa and subpopulations, peristaltic contractions, and the movement of fluid have all been theorized to influence the transport of spermatozoa to the site of fertilization. However, the predominance of each factor is not fully understood. Computational modeling provides a useful method for combining knowledge about the individual processes in complex systems to help understand the relative significance of each factor. The process of constructing and validating an agent-based computational model of sperm movement and transport within the oviductal environment is described in this report. Spermatozoa are modeled as individual cells with a set of behavioral rules defining how they interact with their local environment and regulate their internal state. The inclusion or potential exclusion of each factor is discussed, along with problems identifying parameters and defining behavioral rules from available literature. Finally, the benefits and limitations of the model are described.

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program - Corrected Proof

J.C.F. Jacob, K.T. Haag, G.O. Santos, J.P. Oliveira, M.O. Gastal, E.L. Gastal — 2011-12-26

Abstract: In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to −6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P < 0.03) for flushes performed on Day 6 (42%) compared with all other days. Pregnancy rates for various degrees of synchrony were as follows: +1 (71%), 0 (77%), −1 (68%), −2 (63%), −3 (66%), −4 (76%), −5 (61%), and −6 (27%). The −6 day of degree of synchrony had the lowest (P < 0.05) pregnancy rate compared with all other days, but there was no significant difference among +1 to −5 days. There was a lower (P < 0.05) pregnancy rate for embryos transferred to recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P < 0.05) for Day 7 (76%) embryos compared with Day 6 (50%), Day 8 (64%), and Day 9 (44%) embryos; Day 9 embryos resulted in lower (P < 0.05) pregnancy rates than Days 7 or 8 embryos. In conclusion, this study demonstrated that: (1) embryo recovery rates between Days 7 and 10 were similar and acceptable (e.g., 63% 488/771); (2) the degree of synchrony between donor and recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season.

Doppler sonography of the uterine and ovarian arteries during a superovulatory program in horses - Corrected Proof

M.C. Witt, H. Bollwein, J. Probst, C. Baackmann, E.L. Squires, H. Sieme — 2011-12-23

Abstract: The aim of the present study was to investigate the effects of a gonadotropin treatment to induce superovulation on ovarian and uterine blood flow and its relationship with steroid hormone levels and ovarian response in mares, using color Doppler sonography. Each of six mares were examined sonographically in five cycles for 3 d (t1 to t3) during the follicular development phase (FDP) beginning at a follicle size of ≥ 22 mm, and for 4 d (D-4 to D-1; D0 = Ovulation) in the preovulatory phase (POP). After each examination, total estrogens (Etot) and progesterone (P4) levels were determined in peripheral plasma. Cycles 1, 3, and 5 (c1, c3, c5) were unstimulated cycles (USC); in c2 and c4, the mares were stimulated (SC) with eFSH and inseminated when in estrus at 12 and 24 h after hCG administration. Embryo recovery was performed 6.5 d post ovulation. Cycle 5 c5 was an unstimulated cycle with hCG treatment, insemination, and embryo recovery. Ovarian and uterine blood flow was quantified by the blood flow volume (BFV) and the pulsatility index (PI) in ovarian and uterine arteries. The mean number of ovulations and developing CL was 1.3 ± 0.4 in USC and 4.4 ± 3.1 in stimulated cycles (SC) with no difference (P ≥ 0.05) between the ovaries within mares. No difference (P > 0.05) was observed in utBFV and utPI during FDP between USC and SC, but during POP, utPI was lower (P < 0.05) and utBFV higher (P < 0.001) in SC than USC. The ovBFV was higher (P < 0.01) and ovPI lower (P < 0.05) in SC compared to USC. All uterine and ovarian blood flow parameters were related to the number of developing follicles in SC. Parameters utPI (r = −0.67; P < 0.001) and ovPI (r = −0.53; P < 0.001) were negatively correlated with the number of ovulations on t3, and with the number of collected embryos on t3 (utPI: r = −0.81; P < 0.001), D-4 (utPI: r = −0.64; P < 0.0001), and D-1 (ovPI: r = −0.41; P < 0.01). P4 levels were not positively correlated with utBFV (P > 0.05), but Etot concentrations (D-4: r = 0.790; D3: r = 0.639; P < 0.001; D-1: r = 0.48; P < 0.001) and ovBFV from D-4 to D-1 (r = 0.64; P < 0.001) in SC were. The results of the present study show that in mares treatment with gonadotropins to induce superovulation is associated with a marked increase in uterine and ovarian perfusion, concurrent with the development of multiple follicles and an increase in Etot levels. The increased blood flow seems to be related to the effectiveness of ovarian response to stimulation.

Successful preservation of capercaillie (Tetrao urogallus L.) semen in liquid and frozen states - Corrected Proof

Artur Kowalczyk, Ewa Łukaszewicz, Zenon Rzońca — 2011-12-22

Abstract: Experiments on semen collection and preservation were undertaken by Wrocław University of Environmental and Life Sciences and Forestry Wisła, Poland to assist in the protection of the capercaillie (Tetrao urogallus L.) and to create an ex situ in vitro cryobank. Semen was collected from 11 captive-bred males, using dorsoabdominal massage. Ejaculates once obtained were diluted 3-fold at room temperature with EK diluent and then a number of them were stored at 4 °C for 18, 24, and 48 hours, while the remaining ejaculates were equilibrated with 6% dimethylacetamide and frozen by pipetting, drop-by-drop directly onto a liquid nitrogen surface. Frozen pellets were thawed at 60 °C in a water bath after 4 to 28 mo of storage. In total, 103 individually collected ejaculates (54 stored as liquid and 49 frozen in liquid nitrogen) were of appropriate value for further processing. The volume of ejaculates varied from 30 to 240 μL; spermatozoa concentration from 70 × 106 mL−1 to 1950 × 106 mL−1. The total amount of live spermatozoa in the fresh semen varied from 85.3% to 99.0%, of which from 41.1% to 85.3% were morphologically normal. Among morphologically abnormal forms, bulb-head (5.6% to 36.0%) and midpiece deformations (1.3% to 16.6%) were the most frequent. Dilution and semen storage up to 24 h at 4 °C did not affect the semen quality, as far as motility and sperm morphology are concerned. A significant (P < 0.05) decrease in total live (94.9 vs. 91.7%) and live normal cells (66.4 vs. 56.7%) was observed after 48 h. About 30% to 40% of spermatozoa remained motile. Cryopreservation significantly decreased (P < 0.05) the total number of live and live normal spermatozoa however, in relation to the fresh semen, their average content was 44.1% and 37.4%, respectively. Significant (P < 0.05) individual differences were observed in the quality of the fresh, liquid stored and the frozen-thawed semen assessed in terms of spermatozoa motility and morphology. After a single insemination with thawed semen containing 9.7 million live normal cells, 80% fertility and 100% hatchability were achieved. The obtained results indicate for the first time that there is the potential to use liquid stored and cryopreserved capercaillie semen to support conservation measures for the maintenance of genetic diversity, as well as to increase the number of reintroduced progeny of this endangered grouse species.

“In vitro” capacitation and subsequent acrosome reaction are related to changes in the expression and location of midpiece actin and mitofusin-2 in boar spermatozoa - Corrected Proof

2011-12-22

Abstract: The induction of “in vitro” capacitation (IVC) and subsequent, progesterone-induced “in vitro” acrosome reaction (IVAR) was concomitant with an increase in actin polymerization, also showing an increase in actin presence at the apical area of the midpiece. The presence of mitofusin-2, a protein involved in the regulation of the coordinated mitochondrial function, expanded from midpiece to the principal piece after IVC and IVAR. All of these results indicate that the increase of boar sperm mitochondrial activity during IVC and the first minutes of IVAR is concomitant with changes in the expression and location of both actin and mitofusin-2. Our results suggest that both actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondrial function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure itself.

Permanent contraception of dogs induced with intratesticular injection of a Zinc Gluconate-based solution - Corrected Proof

2011-12-22

Abstract: The objective was to evaluate the efficacy of a single intratesticular injection of a Zinc Gluconate-based solution to induce sterility in male dogs. Fifteen pubertal mongrel dogs (8 mo to 4 y old) were assigned to two groups; Control dogs (n = 5) received a single injection of an isotonic saline solution into each testis, whereas Treated dogs (n = 10), were given Testoblock, a proprietary zinc-gluconate (13.1 mg zinc/ml) solution in a physiological vehicle. The volume of saline or Testoblock injected varied from 0.2 to 1.0 ml/testis (based on testis width). Physical examination, testis width, hematology, clinical chemistry (hepatic and renal function), plasma testosterone concentration, semen characteristics, and libido, were assessed until castration (150 d after treatment). In Treated dogs, testis width increased (approximately 20%) relative to that in Control dogs, but subsequently was not significantly different from Controls (group × time interaction, P < 0.0001). For all dogs, values for hematology and clinical chemistry consistently remained within reference ranges. Although plasma testosterone concentrations decreased over time (P < 0.006), there was only a tendency for an effect of group (P < 0.09), and libido was not significantly affected. By 63 d after Testoblock treatment, eight Treated dogs were azoospermic, whereas the remaining two were oligozoospermic (<10 × 106 sperm/ml). We concluded that intratesticular injection of the Zinc Gluconate-based chemical sterilant Testoblock has considerable potential to induce permanent contraception in male dogs.

Using PGFM (13,14-dihydro-15-keto-prostaglandin F2α) as a non-invasive pregnancy marker for felids - Corrected Proof

M. Dehnhard, C. Finkenwirth, A. Crosier, L. Penfold, J. Ringleb, K. Jewgenow — 2011-12-22

Abstract: Understanding the complex endocrine interactions that control reproduction in felids is essential for captive breeding management. The most important demand is a quick and reliable pregnancy diagnosis. However, the occurrence of pseudopregnancies in felids complicates matters. We investigated whether the fecal prostaglandin metabolite (PGFM) recently suggested for pregnancy diagnosis in the lynx is suitable for all felid species. We found that increased levels of PGFM during the last trimester indicate pregnancy in seven of the eight main lineages of the carnivore family Felidae. PGFM levels in a sand cat (domestic cat lineage) were basal at mating and remained so until Day 40 post-mating. Day 41 marked the beginning of a distinct increase culminating in peak levels of 6.5 μg/g before parturition and decreasing again to baseline thereafter. Similar pregnancy profiles were obtained from the domestic cat, the leopard cat, the lynx, the ocelot and the caracal lineage, whereas in pseudopregnant individuals (sand cat, Iberian and Eurasian lynx) fecal PGFM remained at basal levels. In pregnant cheetahs (puma lineage) PGFM increased above basal following day ∼48 peaking before pregnancy but remained at baseline in pseudopregnant females. Discrepancies existed in the Panthera lineage. While Chinese leopard, Sumatran tiger, and the black panther showed marked increases of PGFM during the last weeks of pregnancy, only moderate increases in PGFM levels were found in the Indochinese tiger and the Persian leopard. Altogether, PGFM as tool for pregnancy diagnosis has been proven to be useful in breeding management of felids.

Sperm concentration at freezing affects post-thaw quality and fertility of ram semen - Corrected Proof

2011-12-22

Abstract: We have investigated the effect of sperm concentration in the freezing doses 200, 400, 800, and 1600 × 106 mL−1 on the post-thaw quality and fertility of ram semen. Semen was collected from seven adult Churra rams by artificial vagina during the breeding season. The semen was diluted in an extender (TES-Tris-fructose, 20% egg yolk, and 4% glycerol), to a final concentration of 200, 400, 800, or 1600 × 106 mL−1 and frozen. Doses were analyzed post-thawing for motility (computer-assisted sperm analysis system [CASA]), viability, and acrosomal status (fluorescence probes propidium iodide [PI]/peanut agglutinin conjugated with fluorescein thiocyanate (PNA-FITC), SYBR-14/PI [Invitrogen; Barcelona, Spain] and YO-PRO-1/PI [Invitrogen; Barcelona, Spain]). Total motility and velocity were lower for 1600 × 106 mL−1 doses, while progressive motility and viability were lower both for 800 and 1600 × 106 mL−1. The proportion of viable spermatozoa showing increased membrane permeability (YO-PRO-1+) rose in 800 and 1200 × 106 mL−1. Intrauterine inseminations were performed with the 200, 400, and 800 × 106 mL−1 doses at a fixed sperm number (25 × 106 per uterine horn) in synchronized ewes. Fertility (lambing rate) was similar for semen frozen at 200 (57.5%) or 400 × 106 mL−1 (54.4%), whereas it was significantly lower for 800 × 106 mL−1 (45.5%). In conclusion, increasing sperm concentration in cryopreserved semen, at least at 800 × 106 mL−1 and more, adversely affects the postthawing quality and fertility of ram semen.

Follicular and endocrinological turnover associated with GnRH induced follicular wave synchronization in Indian crossbred cows - Corrected Proof

2011-12-22

Abstract: The goal of this study was to record the hormonal and follicular turnover in Jersey crossbred cows when subjected for follicular wave synchronization using GnRH. Six healthy, non-lactating and regularly cycling Jersey crossbred cows (5–6 y) were used for the study. In the control group, the follicular wave pattern was ultrasonographically investigated in 18 cycles (3 cycles/cow). In the treatment group, GnRH analogue (buserelin acetate 10 μg im) was administered on Day 6 of the cycle and follicular wave pattern was studied in 12 cycles (2 cycles/animal). Follicular population was categorized based on their diameter Class I, ≤5 mm; Class II, >5–<9 mm; Class III, ≥9 mm) and the number of follicles in each category was determined on Day 6, Day 8 and Day 10. Plasma FSH and progesterone concentrations were estimated in both control and treatment groups. Out of 18 estrous cycles studied, 14 cycles (77.8%), three cycles (16.7%) and one cycle (5.6%) exhibited three-, two- and four-follicular waves per cycle, respectively. It was evident that the DF of Wave I established its dominance and was in the growing phase by Day 6 of the estrous cycle in all the normally cycling crossbred cows. The DF ovulated in all the animals (100%) in the mean interval of 27.7 ± 0.2 h after GnRH administration. A synchronized homogenous group of follicles emerged two days after GnRH injection (Day of 8.0 ± 0.0) in all the animals (100%). The combination of LH surge induced ovulation of DF (abrupt termination of Wave I) and FSH surge stimulated homogenous recruitment of Class I follicles, led to a synchronized emergence of follicular wave. All the GnRH treated cows had three follicular waves because of early emergence and short period of dominance of Wave II DF.

Three-dimensional ultrasound imaging of the equine fetus - Corrected Proof

Y. Kotoyori, N. Yokoo, K. Ito, H. Murase, F. Sato, K. Korosue, Y. Nambo — 2011-12-22

Abstract: The objective was to assess the optimal procedure for real-time, three-dimensional (3D) ultrasound (US) imaging for assessing the equine fetus during the first half of gestation and the possibility of using 3D US imaging of the equine fetus in clinical applications. Seventeen pregnant mares were examined by 3D US between Days 35 and 180 of gestation. Abdominal and endo-vaginal real-time 3D transducers used in human medicine were used for transrectal and transvaginal examinations, respectively. Images were recorded by both 3D stationary and real-time movies. In a comparison of four methods, transrectal examination with a bulb-shaped abdominal 3D transducer enabled the equine fetus to be clearly visualized, and did not require sedation of the mare. Therefore, this approach was the most suitable procedure for examining equine fetuses during the first half of gestation. Each scan required only a few seconds and an entire examination took <10 min in total. The 3D volume image was easy to restore after the examination and could be rotated to any angle the examiner desired. Fetal surface structures, including the head, body, limbs, and genital tubercle, were observed as 3D images which enabled fetal development to be characterized. For early (Days 60–70), but not later (Days 90–150) periods, 3D ultrasonography was not able to evaluate fetal structure in detail as well as conventional 2D ultrasonography. In conclusion, 3D ultrasonography of the equine fetus was a valuable adjunct to 2D ultrasonography and a convenient modality for more detailed assessment of fetal structures.