Theriogenology

Editorial Board

2012-02-01

A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices

A. Bielanski — 2011-09-30

Abstract: This review summarizes pertinent data and opinions regarding the potential hazard of disease transmission through cryopreserved and banked embryos in liquid nitrogen (LN). Special attention is given to the survival of pathogens in LN, new vitrification methods, sterility of LN, risks associated with the use of straws and cryovials, and LN dewars including dry shippers. It was experimentally demonstrated that cross-contamination between LN and embryos may occur, when infectious agents are present in LN and embryos are not protected by a sealed container. It is important, therefore, to prevent direct contact of embryos with LN during cryopreservation and their banking. This includes the usage of hermetically sealed, high-quality, shatter-proof freezing containers and/or the application of a secondary enclosure such as “double bagging or straw in straw.” A periodic disinfection of cryo-dewars should be considered as an additional precaution to diminish the potential for inadvertent cross-contamination. It might be advisable to use separate LN dewars to quarantine embryos derived from infected donors of valuable genotype or from unknown health status, extinction-threatened species. Nevertheless, in summary, it has been concluded that over 25 yr with no direct evidence of disease transmission by transferred cryopreserved human and animal embryos, that the present cryopreservation technology is sanitary sound, with the stipulation that biocontainment measures recommended by the International Embryo Transfer Society (IETS) and the World Organization for Animal Health - Office International des Epizooties (OIE), are strictly followed.

Slow-controlled freezing versus speed-cooling for cryopreservation of whole guinea pig ovaries

2011-09-30

Abstract: The objective of this study was to evaluate the feasibility of whole-ovary perfusion, and to compare the effects of speed-cooling and slow-controlled freezing of whole guinea pig ovaries. Slow-freezing and speed-cooling procedures were performed after perfusion of guinea pig ovaries with cryoprotectants. Ink perfused via the vascular pedicles was present in the microvessels around various follicles at various stages of development in the cortical and medullar regions, thereby confirming that perfusion was effective. Vascular damage was essentially confined to the cannulated artery. Based on histological examination, there were (mean ± SEM) 93.1 ± 4.2, 79.0 ± 2.0, and 54.7 ± 8.5% healthy follicles in the fresh, slow-freezing and speed-cooling groups, respectively (each group differed from the other two, P < 0.05). Trypan blue staining of isolated follicles confirmed that cellular damage was greater following speed-cooling than slow-freezing (58.6 vs 29.2%, P < 0.05). Based on a TUNEL assay, speed-cooling caused more apoptotic granulosa and theca cells in antral follicles than slow-freezing. In conclusion, the present study provided evidence that guinea pig whole ovaries could be perfused with cryoprotectant and cryopreserved in vitro. Furthermore, the slow-freezing protocol resulted in less cellular damage in thawed tissues than speed-cooling.

Induction of PGFM pulses and luteolysis by sequential estradiol-17β treatments in heifers

G. Pugliesi, M.A. Beg, G.R. Carvalho, O.J. Ginther — 2011-11-28

Abstract: The effects of sequential induction of PGFM pulses by estradiol-17β (E2) on prominence of PGFM pulses and progesterone (P4) concentration were studied in heifers. Three treatments of vehicle (n = 12) or E2 (n = 12) at doses of 0.05 or 0.1 mg were given at 12-h intervals beginning on Day 15 postovulation. Blood samples were collected every 12 h from Days 13–24 and hourly for 12 h after the first and third treatments. On Day 15, all heifers were in preluteolysis and on Day 16 were in preluteolysis in the vehicle-treated heifers (n = 11) and either preluteolysis (n = 4) or luteolysis (n = 8) in the E2-treated heifers. Peak concentration of induced PGFM pulses during preluteolysis on Day 15 was greater (P < 0.04) than for pulses during preluteolysis on Day 16. The interval from ovulation to the beginning of luteolysis was shorter (P < 0.04) in the E2-treated heifers than in the vehicle-treated heifers. An E2-induced PGFM pulse was less prominent (P < 0.008) in heifers in temporal association with a transient resurgence in P4 than in heifers with a progressive P4 decrease. The hypothesis that repeated E2 exposure stimulates increasing prominence of PGFM pulses was not supported. Instead, repeated exposure reduced the prominence of PGFM pulses, in contrast to the stimulation from the first E2 treatment. Reduced prominence of a PGF2α pulse during luteolysis can lead to a transient resurgence in P4 concentration.

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

Artur Kowalczyk, Ewa Łukaszewicz — 2011-10-19

Abstract: The objective of the present experiments was to produce the intergeneric hybrids of domesticated and wild goose via artificial insemination with fresh and frozen-thawed semen. The experiments were carried out during two successive goose reproductive seasons, on eight five-year-old Canada Goose (Branta canadensis L.) males used as semen donors and 16 two-year-old White Kołuda geese designated to fertility tests. Pooled semen was collected twice a week by the dorso-abdominal massage. In freshly collected semen, ejaculate volume, color, consistency, degree of fecal or blood contamination, spermatozoa concentration, motility, and morphology were evaluated. Part of the semen collected in the first year of the experiment (Experiment 1) was used for geese insemination with fresh semen, while the remainder was frozen. In Experiment 2 all samples were subjected exclusively to freezing procedure. Geese were inseminated once a week with fresh semen in a dose of 80 μl or 160 μl, and twice a week with frozen-thawed semen in a dose of 80 μl (160 μl per wk) or 100 μl (200 μl per wk). Eggs were set weekly and incubated up to hatching. The volume of ejaculates varied from 0.100 to 0.470 ml; spermatozoa concentration from 140 to 310 million ml−1; progressive movement was observed in 40 to 60% of spermatozoa; the percentage of total live spermatozoa ranged from 69.3 to 92.0%, the highest percentage (34.0–68.3) was represented by live normal spermatozoa and those with bulb-head (13.3–41.0). Cryopreservation caused a decrease in percentage of motile cells to 30%; total live spermatozoa contribution by 27.2%p, including those live normal by 15.9%p (in relation to the fresh semen), bulb-head spermatozoa by 10.9%p, and increase (by 5.9%p) in number of spermatozoa with other deformations. Goose insemination 1×/week with fresh semen containing about 10.3 million live normal spermatozoa resulted in 66.7% of fertile eggs and with dose higher by 2.8 million spermatozoa (on average) the fertility increased by 20.9%p (up to 87.6% on average). Hatchability from set and fertile eggs was 55.9% and 83.9% vs. 66.3% and 75.6%, respectively. After twice a week insemination with frozen-thawed semen containing about 10.2 million live normal cells 58.2% eggs were fertile; hatchability from set eggs was 42.8% and from fertile eggs 71.7%, while insemination dose increase by 2.7 million spermatozoa per week caused a fertilization increase by 3.8%p (62.0% on average), this increase was not statistically significant, but hatchability from the fertile eggs (95.4%), was significantly (P < 0.05) higher. The use of AI with fresh semen in the creation of intergeneric hybrids of Canada goose males and White Kołuda females allows a high level of egg fertility to be obtained. Furthermore, one limitation which is the short reproductive season of the Canada goose may be overcome by the use of cryopreserved semen.

Ultrasound characteristics of experimentally induced luteinized unruptured follicles (LUF) and naturally occurring hemorrhagic anovulatory follicles (HAF) in the mare

J. Cuervo-Arango, J.R. Newcombe — 2011-09-30

Abstract: The development of hemorrhagic anovulatory follicles (HAF) involves luteinization and hemorrhage of the follicle. This is observed on ultrasound as an increase in the echogenicity of the granulosa layer and formation of echoic particles in the antrum. The inhibition of prostaglandin synthesis with flunixin meglumine (FM) during the periovulatory period induces ovulatory failure with development of luteinized unruptured follicles (LUF). These two types of anovulatory follicles appear to share similar ultrasound features but they have not been compared critically. The following endpoints: follicle diameter, follicular contents score, interval from hCG administration to beginning of follicular hemorrhage, interval from hemorrhage to organization of follicular contents, and cycle length were studied and compared in mares with HAF (n = 11) and LUF (n = 13). The objective of this study was to elucidate whether these two unruptured follicles have a consistent clinical pattern of development and therefore can be considered as part of the same anovulatory syndrome. None of the endpoints analyzed differed significantly between HAF and LUF. However, there was a greater individual variation in HAF as compared with LUF in regards to interval from hCG to hemorrhage, follicular diameter at the administration of hCG, and beginning of hemorrhage. In conclusion, HAF share a similar cascade of ultrasound characteristics with the experimentally induced LUF. This finding may provide new insights in elucidating the pathogenesis of HAF.

Can video cameras replace visual estrus detection in dairy cows?

2011-12-05

Abstract: A 6-mo experiment was conducted in a dairy herd to evaluate a video system for estrus detection. From October 2007 to April 2008, 35 dairy cows of three breeds that ranged in age from 2 to 6 yr were included in the study. Four daylight cameras were set up in two free stalls with straw litter and connected to a computer equipped with specific software to detect movement. This system allowed the continuous observation of the cows as well as video storage. An observation method related to the functionality of the video management software (“Camera-Icons” method) was used to detect the standing mount position and was compared to direct visual observation (direct visual method). Both methods were based on the visualization of standing mount position. A group of profile photos consisting of the full face, left side, right side, and back of each cow was used to identify animals on the videos. Milk progesterone profiles allowed the determination of ovulatory periods (reference method), and a total of 84 ovulatory periods were used. Data obtained by direct visual estrus detection were used as a control. Excluding the first postpartum ovulatory periods, the “Camera-Icons” method allowed the detection of 80% of the ovulatory periods versus 68.6% with the direct visual method (control) (P = 0.07). Consequently, the “Camera-Icons” method gave at least similar results to the direct visual method. When combining the two methods, the detection rate was 88.6%, which was significantly higher than the detection rate allowed by the direct visual method (P < 0.0005). Eight to 32 min (mean 20 min) were used daily to analyze stored images. When compared with the 40 min (four periods of 10 min) dedicated to the direct visual method, we conclude that the video survey system not only saved time but also can replace direct visual estrus detection.

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer

C.E. Pope, M.C. Gómez, N. Kagawa, M. Kuwayama, S.P. Leibo, B.L. Dresser — 2011-10-19

Abstract: We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients.

Sequence analysis of feline oviductin and its expression during the estrous cycle in the domestic cat (Felis catus)

A. Hachen, K. Jewgenow, B.C. Braun — 2011-10-19

Abstract: Oviductins belong to a family of oviduct-specific glycoproteins believed to play an important role in fertilization and/or early embryonic development. Oviductin cDNA between species is highly conserved and shares 58% to 98% similarity in the deduced amino acid sequences. Our objective in this study was to sequence the full open reading frame of the feline oviductin and to examine its expression during the estrous cycle on both mRNA and protein level. The obtained cDNA containing the full open reading frame was determined to be 1677 nucleotides coding for a deduced protein of 558 amino acids. Identities between species range from 74% (mouse) to 80% (human, baboon, and rhesus) within the N-terminal protein region. Major differences were localized in the carboxy terminal region, which corresponds to exon 11 of the gene. Feline oviductin contained one putative N-linked glycosylation site, six O-linked glycosylation sites, a potential heparin binding site, and two cholesterol recognition and/or interaction amino acid consensus (CRAC) domains. Oviductin expression was analyzed by real time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. Both approaches revealed an estrous cycle-dependent expression in the ampulla and isthmus. Quantitative PCR showed highest oviductin mRNA copy numbers in the early and late follicular stage and reduced mRNA expression during all other stages. With the exception of the early follicular stage, feline oviductin mRNA abundance was not significantly different in the oviductal segments ampulla and isthmus. A prominent immunolabeling was seen in the early and late follicular stage which disappeared after ovulation, indicating a function of the protein during sperm storage and fertilization.

Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: Changes with age and effects of cryptorchidism

2011-10-19

Abstract: The objectives were to: (1) develop a time-resolved fluorescence immunoassay (TRFIA) to measure insulin-like peptide 3 (INSL3) in canine plasma; (2) investigate changes of plasma concentrations of INSL3 and testosterone with age in normal male dogs; and (3) compare hormonal concentrations among cryptorchid, normal, and castrated dogs to evaluate endocrine function of the Leydig cell component in retained testes. Blood samples were taken from normal male dogs from prepubertal age to advanced age (4 mo to 14 y, n = 89), and from unilateral cryptorchid (n = 31), bilateral cryptorchid (n = 7), and castrated dogs (n = 3). Canine plasma INSL3 was measured with a newly developed TRFIA. The minimum detection limit of the INSL3 assay was 0.02 ng/ml and the detection range was 0.02 to 20 ng/ml. Plasma INSL3 concentrations increased (P < 0.05) from prepubertal age (4–6 mo) to pubertal age (6–12 mo), and then declined (P < 0.05) from pubertal age to post-pubertal age (1–5 y), reaching a plateau. Plasma testosterone concentrations increased (P < 0.0001) dramatically from prepubertal to pubertal ages, and then seemed to plateau. Concentrations of both INSL3 and testosterone were lower (P < 0.0001 for each) in bilateral cryptorchid dogs than in normal and unilateral cryptorchid dogs. The INSL3 (range: 0.05–0.43 ng/ml) and testosterone (range: 0.10–0.94 ng/ml) concentrations were readily detected in bilateral cryptorchids, but not in castrated dogs (INSL3 < 0.02 ng/ml; testosterone < 0.04 ng/ml). In conclusion, plasma INSL3 concentrations in male dogs measured by a newly developed TRFIA had a transient surge at a pubertal age, whereas testosterone did not. Lower plasma concentrations of INSL3 and testosterone in bilateral cryptorchid dogs suggest impaired endocrine functions of Leydig cell component in paired retained testes. Therefore, peripheral plasma INSL3 and testosterone concentrations have potential diagnostic value in predicting the presence of bilaterally retained testes in male dogs.

Amino acids in cat fallopian tube and follicular fluids

P. Guérin, E. Rosset, M. Rey, G. Febvay, P. Bruyère, N. Corrao, V. Neto, S. Buff — 2011-10-07

Abstract: Aminograms of tubal and follicular fluids were obtained using fluids collected by aspiratory puncture from six cats. The amino acids were separated and quantified by high-performance liquid chromatography analysis. The serum of the cats was used as control. The three most prevalent amino acids quantified in cat tubal fluid were glycine, glutamic acid, and taurine. Their mean concentrations were 840 μmol/l (μm), 808 μm and 596 μm, respectively. The three most prevalent amino acids quantified in cat follicular fluid were alanine, glutamine, and taurine. Their mean concentrations were 359 μm, 351 μm, and 258 μm, respectively. This result is consistent with aminograms of tubal fluid previously determined in other mammals. As previously observed in other species and humans, glycine was quantitatively the most abundant and most prevalent free amino acid in cat tubal fluid. The total quantity of amino acids in tubal fluid was similar in cats and other species. However, in contrast with other species studied, hypotaurine was not detected in tubal and follicular fluids of female cats.

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome

D. Panzani, A. Rota, A. Crisci, H. Kindahl, N. Govoni, F. Camillo — 2011-11-07

Abstract: Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF2α release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0–3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results.

Effects of polymorphonuclear neutrophile infiltration into the endometrial environment on embryonic development in superovulated cows

M. Drillich, D. Tesfaye, F. Rings, K. Schellander, W. Heuwieser, M. Hoelker — 2011-11-07

Abstract: Recent studies on bovine uterine disorders have demonstrated that endometrial infiltration with polymorphonuclear neutrophils (PMN) in the postpartum period or at the time of breeding negatively affects reproductive performance. The objective of the present study was therefore to analyze the effect of endometrial PMN infiltration on superovulation outcome. Cows were synchronized and superovulated receiving a total of three artificial inseminations within 24 h. Endometrial cytologic samples were collected by cytobrush technique at first artificial inseminations (AI) (d −1) and before embryo flush (d 7). Embryos were recovered by uterus flushing at Day 7 and evaluated for total cell number and apoptotic cell index. A total of 425 embryos were flushed out of 48 superovulated cows. The PMN dynamics from first AI to flushing had a significant effect on flushing outcome. Significant differences in terms of number of palpable corpora lutea (14.1 vs 7.2) and transferable embryos (8.8 vs 1.9) were found between cows with PMN proportions increasing from zero (0%) at AI to positive proportions (> 0%) at flushing (group PMNZP) and cows with higher endometrial PMN proportions decreasing to lower but still positive proportions from AI to flushing (group PMNHL). Moreover, cows classified to PMN class zero at first AI flushed a significant higher number of total embryos (10.3 vs 6.9) and transferable embryos (6.8 vs 3.7) compared to cows of PMN class positive at first AI (P > 0.05) in our study. Considering a significant interaction effect between PMN class at first AI and flush (P < 0.05), PMN class at first AI (d −1) correlated significantly with number of total flushed and transferable embryos only in combination with a positive PMN class at flush (d 7). Likewise, PMN class at flush (d 7) beard a significant effect on total number of flushed embryos only when classified to PMN class zero at first AI. Collectively, the present work is the first study that demonstrated a significant relationship between endometrial PMN infiltration at first AI as well as PMN dynamic from first AI to time of flush and superovulation outcome.

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus)

2011-11-07

Abstract: Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag–NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag–NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method

A. Kumaresan, A. Johannisson, F. Saravia, A.S. Bergqvist — 2011-10-07

Abstract: Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO2. Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.

Toxicity of cryoprotectants to honey bee semen and queens

J. Wegener, K. Bienefeld — 2011-11-24

Abstract: Given the threats to the intraspecific biodiversity of Apis mellifera and the pressure on bee breeding to come up with disease-tolerant lines, techniques to cryopreserve drone semen are of great interest. Freeze-thawed drone semen of high viability and/or motility has repeatedly been obtained, but fertility of such semen, when it was measured, was always low. The cryoprotective agent (CPA) most frequently used with drone semen is dimethyl sulfoxide (DMSO), although this substance has been suspected of causing genetic damage in sperm. No form of sperm washing is currently performed. Using a membrane permeability assay, we measured the short-term toxicity of four possible replacements for DMSO, 1,3-propane diol, 2,3-butane diol, ethylene glycol, and dimethyl formamide. We also tested whether the practice of inseminating queens with CPA-containing semen affects sperm numbers in the storage organs of queens, or sperm fertility. Finally, we tested whether CPA-toxicity in vivo can be reduced by using mixtures of two CPAs, DMSO, and ethylene glycol. Our results show that, although short-term toxicity of all CPAs tested was low, the presence of single CPAs in insemination mixtures at concentrations required for slow freezing greatly reduced the number of sperm reaching the spermatheca. Contrary to earlier reports, this was also true for DMSO. Ethylene glycol was additionally shown to reduce the viability of spermatozoa reaching the storage organ. Mixtures of DMSO and EthGly performed better than either substance used singly at the same concentration. We conclude that the toxicity of CPAs, including DMSO, on honey bee semen and/or queens has been underestimated in the past. This could partly explain the discrepancy between in vitro and in vivo quality of cryopreserved drone semen, described by others. Combinations of several CPAs and techniques to partly remove CPAs after thawing could help to solve this problem.

Expression of vascular endothelial growth factor (VEGF) transcript and protein in the testis of several vertebrates, including endangered species

2011-11-07

Abstract: Vascular endothelial growth factor (VEGF) is known to influence the testis function. To establish the role of VEGF in the testis of a variety of species, we analyzed the expression of VEGF transcript using human gene-specific primers by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis in the testes of 18 vertebrates, including a few endangered species. An amplicon of 566 bp representing VEGF165 was identified in testis of all species in this study. Sequence analysis of these amplicons revealed 84 to 96% homology to available human VEGF sequence and to the VEGF sequences of other species in GenBank. Immunohistochemical analysis revealed expression of VEGF protein, primarily in Sertoli and Leydig cells and occasionally in the germ cells of the testis sections. It can be concluded from this study that expression of VEGF transcript is conserved in the testis of several vertebrates and may have a role in the process of spermatogenesis.

Influence of testicular hormones on the somatostatin-GH system during the growth promoted transition to puberty in sheep

Marta Wańkowska — 2011-11-07

Abstract: The aim of the present study was to investigate whether the growth promoted transition to puberty in lambs involved changes in the effects of testicular hormones on somatostatin in hypothalamic neurons and GH secretion. The study was performed in infants (9-week-old) testis-intact (TEI) and orchidectomized (ORCHX) at the sixth week of age, and pubertal lambs (16-week-old) TEI and ORCHX at the 12th week of age (n = 20). In TEI lambs, the changes included a pubertal increase in immunoreactive somatostatin in the periventricular nucleus and median eminence with simultaneous neuropeptide depletion in the median eminence, and a decrease in the percentage of the hypophyseal area (PA) occupied by GH-immunoreactive cells (P < 0.05). The mean concentration of GH in the peripheral blood plasma was greater (P < 0.001) in early infancy (5 wk), because of the greater (P < 0.0001) pulse amplitude, and then uniformly low until puberty. The postnatal increase in the body weight (BW) was prominent (P < 0.01) in middle-late infancy (9–12 wk) because of the large daily live-weight gain. After orchidectomy somatostatin was abundant. This effect on nerve terminals in the median eminence was greater (P < 0.01) in infancy and lesser (P < 0.05) in puberty. Conversely, the PA occupied by GH cells was lower in the ORCHX pubertal lambs compared to TEI lambs (P < 0.05). The GH concentration and pulse characteristics were less (P < 0.05) in the infantile and pubertal ORCHX lambs compared to the TEI lambs. However, this effect was weak (P < 0.05) until middle infancy because of no influence on the GH basal concentration, and strong (P < 0.001) after late infancy. The BW did not differ (P > 0.05) between TEI and ORCHX lambs. Findings suggest activation of GH negative autofeedback loop in middle infancy. Testicular factors may play an inhibitory role in regulating somatostatin accumulation and a stimulatory role in GH secretion until puberty. The start of puberty is related to an attenuation in the stimulatory role of gonadal factors in regulating somatostatin depletion in nerve terminals associated with an intensification of the stimulatory role of gonadal factors in regulating GH secretion. From a somatic perspective of growth rate, these mechanisms do not seem to be important. Thus, testicular factors modulate mechanisms within the somatostatin-GH system to integrate somatotropic and gonadotropic functions at the time of growth-promoted sexual maturation in sheep.

Comparison of assessment of pigeon sperm viability by contrast-phase microscope (eosin-nigrosin staining) and flow cytometry (SYBR-14/propidium iodide (PI) staining) [evaluation of pigeon sperm viability]

M.D. Klimowicz-Bodys, F. Batkowski, A.S. Ochrem, M.A. Savič — 2011-11-07

Abstract: The aim of these experiments was to compare the conventional, microscopic method of evaluating pigeon sperm viability to sperm assessed by flow cytometry. Semen was collected twice a week from two groups of pigeons. In every group were 20 males (Group I: meat-type breed; Group II: fancy pigeon breed). Semen was collected using the lumbosacral and cloacal region massage method. Ejaculates collected from each group were pooled and diluted to 10 × 106 sperm/ml in BPSE solution. Samples were divided into three equal parts and estimated after collection as well as after in vitro storage for 3, 6 and 24 h. The first part was using for semen motility evaluation. The proportion of motile spermatozoa (MOT) and progressive movement (PMOT) of fresh and stored semen were evaluated using the CASA-system. The second part was examined subjectively by microscope (eosin-nigrosin (EN), eosin-nigrosin staining), the third one was assessed using dual fluorescence SYBR-14/propidium iodide (PI) and flow cytometry (FC). There were not any significant differences in sperm viability and motility between the groups at 0, 3, 6, and 24 h post collection. The percentage of viable spermatozoa in fresh semen determined by EN and FC was not different in Groups I and II (I - 88.71 ± 5.42 and 84.01 ± 3.19, respectively; II-90.87 ± 6.01 and 87.38 ± 5.57, respectively). Significantly lower percentages of viable spermatozoa were detected by FC compared to the EN method in both groups after 6 h (P ≤ 0.05) as well as 24 h (P ≤ 0.01) of storage. Moreover, the dual fluorescent SYBR-14/PI staining allowed for the identification a third population of double stained, moribund spermatozoa. High positive correlations in percentage of live spermatozoa were noted between EN and FC methods in both groups of birds. Evaluation of sperm viability by FC is a rapid, accurate, sensitive, and objective method for the assessment of pigeon sperm viability in fresh as well as stored semen.

The expression and putative role of brain-derived neurotrophic factor and its receptor in bovine sperm

C. Li, C. Li, X. Zhu, C. Wang, Zhuo Liu, W. Li, Chen Lu, Xu Zhou — 2011-10-19

Abstract: The neurotrophin family of proteins promote the survival and differentiation of nerve cells and are thought to play an important role in development of reproductive tissues. The objective of the present study was to detect the presence of Brain-derived neurotrophic factor (BDNF) and its receptor TrkB in bovine sperm, and explore the potential role of BDNF in sperm function. We demonstrated that both the neorotrophin BDNF and the tyrosine kinase receptor protein TrkB were expressed in ejaculated bovine sperm. Furthermore, BDNF per se was secreted by sperm. Insulin and leptin secretion by bovine sperm were increased (P < 0.01) when cells were exposed to exogenous BDNF, whereas insulin was decreased by K252a. Therefore, we inferred that BDNF could be a regulator of sperm secretion of insulin and leptin through the TrkB receptor. Sperm viability and mitochondrial activity were both decreased (P < 0.05) when the BDNF/TrkB signaling pathway was blocked with K252a. Furthermore, BDNF promoted apoptosis of bovine sperm through TrkB binding (P < 0.05). In conclusion, these observations provided evidence that BDNF secreted by bovine sperm was important in regulation of insulin and leptin secretion in ejaculated bovine sperm. Furthermore, BDNF may affect sperm mitochondrial activity and apoptosis, as well as their viability.

Can caprine arthritis encephalitis virus (CAEV) be transmitted by in vitro fertilization with experimentally infected sperm?

2011-10-19

Abstract: For each of the five fertilization trials of the experiment, frozen semen was prepared for in vitro capacitation at a concentration of 1 × 107 spz/ml and divided into three groups. One group was used as a control, while the two others were inoculated with 100 μl/ml of either culture medium from non-infected cells (placebo group) or cell culture medium containing virus at a concentration of 105 TCID50/ml (infected group). A total of 789 oocytes were used for IVF. For each of the five trials a group of oocytes were used as a non-infected control and were found to be caprine arthritis-encephalitis virus (CAEV) free. The other oocytes were divided in two equal batches. Oocytes in the first batch were in vitro fertilized with CAEV infected sperm (infected group) and the second batch were fertilized with CAEV non-infected sperm (placebo and control groups). After IVF, the zygotes of each group were washed 12 times. The CAEV genome was not detected (using RT-PCR) in the washing media of either the control or placebo groups from each trial. In contrast, the first three washing media from the infected group were consistently found to be positive for the CAEV genome (5/5), whereas subsequent washing media were CAEV-free (P < 0.05). Zygotes obtained using all semen groups tested negative for both the provirus and genome of CAEV. These results clearly show that the first four washes were sufficient to remove viral particles from CAEV infected fertilization media and that CAEV-free embryos can be produced by IVF using spermatozoa infected in vitro by CAEV.

Reproductive seasonality and the effect of the GnRH agonist deslorelin as a contraceptive in captive male Black Flying-foxes (Pteropus alecto)

2011-11-24

Abstract: Effective contraception would enhance genetic management of captive Pteropus species, which typically breed well in captivity. Male reproductive seasonality was monitored (15-mo interval) in captive P. alecto (6 controls and 5 treated with 4.7 mg deslorelin). In untreated males, there were seasonal changes in testicular volume, body weight and testosterone secretion; testicular volume and body weight peaked in February and March, respectively, whereas testosterone concentration remained >5 ng/ml before rising (P < 0.001) to 24.9 ± 3.6 ng/ml (mean ± SEM) in April. However, there was no corresponding change in sperm quality, and seminal vesicle gland (SVG) secretions remained present in ejaculates. In treated males, testosterone concentration had an initial ‘flare’ response (mean ± SEM peak: 19.95 ± 3.27 ng/ml) before declining (P < 0.001) by 32 d to basal levels, where it remained. In these males, there was reduced sperm motility after 1 mo (P < 0.001) and the absence of SVG secretions after 4 mo. However, aspermic ejaculates were first recorded 5 mo post-treatment. At 10 mo after treatment, spermatogenesis was still disrupted, when membrane-intact, but non-motile sperm were present in two individuals. Motile sperm were first recovered from one of these males 13 mo after deslorelin treatment. We concluded that captive P. alecto males: (a) had seasonal reproductive changes in testicular volume, body weight and testosterone secretion; (b) produced motile, membrane-intact sperm and SVG secretions throughout the year; and (c) had a rapid decline in testosterone concentration and consequent suppression of testicular function for at least 5 mo following deslorelin administration.

Effects of the GnRH analogue deslorelin implants on reproduction in female domestic cats

T.S.F. Toydemir, M.R. Kılıçarslan, V. Olgaç — 2011-10-19

Abstract: The aim of the present study was to investigate the safety and efficacy of deslorelin, a GnRH agonist, implants in suppressing estrus behavior and matings in a controlled ambient environment in feline queens in the presence of a tomcat. Local and utero-ovarian side effects of deslorelin implants were also investigated. The queens were housed in groups and assigned to one of three treatments: group 1 received 9.5 mg deslorelin implants (N = 14), group 2 received 5 mg megestrol acetate tablets and 9.5 mg deslorelin implants (N = 7), and group 3 were given placebo implants (N = 7). All implants were placed subcutaneously cranial to the interscapular region under xylazine hydrochloride sedation. Ovarian activity was monitored by fecal estradiol (E2) analyses. The animals were observed daily and checked individually at three-day intervals for behavioral signs of estrus. After 18.5 mo of trial, queens were ovariohysterectomized, and ovaries and uteri were weighed and evaluated histologically. E2 levels were significantly lower in group 1 and 2 than in group 3 with an average of 128.48 ± 19.97 ng/g, 90.44 ± 7.16 ng/g and 283.26 ± 39.21 ng/g, respectively, excepting the first week of treatment. After inserting implants an initial estrus-like increase in fecal E2 concentrations occurred in all treated queens except one female in group 2. Ovarian and uterine weights were significantly different among the groups (P < 0.01), and were lowest in groups 1 and 2. Primordial and primary follicle numbers were significantly higher in groups 1 and 2 than in group 3 (P < 0.001). Endometrial gland, antral follicle, and corpus luteum (CL) numbers were highest in group 3 (P < 0.01, 0.001, and 0.001, respectively) compared with groups 1 and 2. Deslorelin implants successfully suppressed estrus behavior and E2 secretion in queens for 18.5 mo of the study period. Further investigations are needed to demonstrate the effects of GnRH agonists on ovarian interstitial tissue.

Similar rates of chromosomal aberrant secondary oocytes in two indigenous cattle (Bos taurus) breeds as determined by dual-color FISH

2011-11-04

Abstract: In vitro-matured metaphase II (MII) oocytes with corresponding first polar bodies (I pb) from two indigenous cattle (Bos taurus) breeds have been investigated to provide specific data upon the incidence of aneuploidy. A total of 165 and 140 in vitro-matured MII oocytes of the Podolian (PO) and Maremmana (MA) breeds, respectively, were analyzed by fluorescence in situ hybridization using Xcen and five chromosome-specific painting probes. Oocytes with unreduced chromosome number were 13.3% and 6.4% in the two breeds, respectively, averaging 10.2%. In the PO, out of 100 MII oocytes + I pb analyzed, two oocytes were nullisomic for chromosome 5 (2.0%) and one disomic for the same chromosome (1.0%). In the MA, out of 100 MII oocytes + I pb, one oocyte was found nullisomic for chromosome 5 (1.0%) and one was disomic for the X chromosome (1.0%). Out of 200 MII oocytes + I pb, the mean rate of aneuploidy (nullisomy + disomy) for the two chromosomes scored was 2.5%, of which 1.5% was due to nullisomy and 1.0% due to disomy. By averaging these data with those previously reported on dairy cattle, the overall incidence of aneuploidy in cattle, as a species, was 2.25%, of which 1.25% was due to nullisomy and 1.0% due to disomy. The results so far achieved indicate similar rates of aneuploidy among the four cattle breeds investigated. Interspecific comparison between cattle (Xcen-5 probes) and pig (Sus scrofa domestica) (1–10 probes) also reveal similar rates. Further studies are needed that use more probes to investigate the interchromosomal effect. Establishing a baseline level of aneuploidy for each species/breed could also be useful for improving the in vitro production of embryos destined to the embryo transfer industry as well as for monitoring future trends of the reproductive health of domestic animals in relation to management errors and/or environmental hazards.

The expression of genes encoding zona pellucida glycoproteins in canine cumulus-oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol

2011-11-24

Abstract: The role of progesterone (P4) and estradiol-17beta (E2) on the efficiency of canine oocyte maturation in vitro is recognized, but little is known about the influence of both steroids on the expression of zona pellucida (ZP) glycoproteins. It has been shown that E2 and P4 used in the IVC significantly influenced canine oocytes meiotic competence, although the effect is specifically related to the combination of hormones used in the experiment. Because both of these steroids may stimulate or inhibit maturation competence of oocytes in a dose-dependent manner, there is a high possibility that they also influence the fertilization ability of canine oocytes. Our study was aimed to analyze whether genes, encoding ZP glycoproteins, are regulated by P4 or E2. Canine cumulus oocyte complexes (COCs) were recovered from anestrous mongrel bitches after ovariohysterectomy and cultured in serum-free tissue culture medium 199. The expression pattern of ZP glycoproteins 2 and 3 (ZP2 and ZP3) mRNAs, using quantitative real-time polymerase chain reaction (RQ-PCR), and of ZP3 and ZP4 proteins, using Western blot analyses, was examined in oocytes after the supplementation of the culture medium with (1) 0.5 μg/mL, 1.0 μg/mL, and 2.0 μg/mL of P4 (experiment 1), or with (2) 2.0 μg/mL E2, and with (3) a combination of E2 (2.0 μg/mL) and P4 (0.5, 1.0, or 2.0 μg/mL, respectively; experiment 2). The analysis revealed an inhibited expression of ZP2 mRNA in oocytes after in vitro maturation (IVM) with different P4 supplementations as compared with oocytes before IVM. The expression of ZP3 mRNA was stimulated (P < 0.01) by the supplementation of 1.0 μg/mL P4. The expression of both ZP3 and ZP4 proteins was also stimulated after the treatment with 1.0 μg/mL P4. On the other hand, the level of ZP2 mRNA was inhibited (P < 0.01) after the supplementation with E2 or with combinations of E2 and P4 as compared with control oocytes. The expression of ZP3 mRNA was significantly higher after the supplementation with E2 and 0.5 μg/mL P4. Similarly, ZP3 and ZP4 proteins were highly expressed (P < 0.01) after such hormone supplementation. The results clearly show that in vitro, P4 regulates the expression of ZP glycoproteins in a dose-dependent manner. We demonstrated that E2 used alone and in combination with P4 upregulates the expression of ZP3 mRNA as well as ZP3 and ZP4 protein in canine oocytes. ZP2 mRNA is downregulated by E2 alone and in combination with E2 and P4. Furthermore, ZP glycoproteins expression is regulated by E2 alone or in combination with P4, and such synergistic or adverse effect is P4 concentration-dependent.

Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA)

2011-10-17

Abstract: In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5–13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.

Announcement

2012-02-01

February 2012 Announcement and Call for Abstracts

Theriogenology

Editorial Board

Contents

A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices

Slow-controlled freezing versus speed-cooling for cryopreservation of whole guinea pig ovaries

Induction of PGFM pulses and luteolysis by sequential estradiol-17β treatments in heifers

The possibility of obtaining intergeneric hybrids via White Kołuda (Anser anser L.) goose insemination with fresh and frozen-thawed Canada goose (Branta canadensis L.) gander semen

Ultrasound characteristics of experimentally induced luteinized unruptured follicles (LUF) and naturally occurring hemorrhagic anovulatory follicles (HAF) in the mare

Can video cameras replace visual estrus detection in dairy cows?

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer

Sequence analysis of feline oviductin and its expression during the estrous cycle in the domestic cat (Felis catus)

Plasma insulin-like peptide 3 and testosterone concentrations in male dogs: Changes with age and effects of cryptorchidism

Amino acids in cat fallopian tube and follicular fluids

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome

Effects of polymorphonuclear neutrophile infiltration into the endometrial environment on embryonic development in superovulated cows

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus)

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method

Toxicity of cryoprotectants to honey bee semen and queens

Expression of vascular endothelial growth factor (VEGF) transcript and protein in the testis of several vertebrates, including endangered species

Influence of testicular hormones on the somatostatin-GH system during the growth promoted transition to puberty in sheep

Comparison of assessment of pigeon sperm viability by contrast-phase microscope (eosin-nigrosin staining) and flow cytometry (SYBR-14/propidium iodide (PI) staining) [evaluation of pigeon sperm viability]

The expression and putative role of brain-derived neurotrophic factor and its receptor in bovine sperm

Can caprine arthritis encephalitis virus (CAEV) be transmitted by in vitro fertilization with experimentally infected sperm?

Reproductive seasonality and the effect of the GnRH agonist deslorelin as a contraceptive in captive male Black Flying-foxes (Pteropus alecto)

Effects of the GnRH analogue deslorelin implants on reproduction in female domestic cats

Similar rates of chromosomal aberrant secondary oocytes in two indigenous cattle (Bos taurus) breeds as determined by dual-color FISH

The expression of genes encoding zona pellucida glycoproteins in canine cumulus-oocyte complexes cultured in vitro in media supplemented with progesterone and estradiol

Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA)

Announcement