Theriogenology - Articles in Press

Sonic Hedgehog improves in vitro development of porcine parthenotes and handmade cloned embryos - Corrected Proof

2010-07-21

Abstract: This study investigated the expression of Sonic Hedgehog (Shh) signaling pathway and its effect on porcine parthenogenetic (PA) embryo development. The Shh receptor Patched (Ptc1) and co-receptor Smoothened (Smo) were expressed at various stages of PA porcine embryos, at both mRNA and protein levels. Furthermore, the transcriptional activator Gli1 mRNA was first present in the 2-cell stage embryos, and was readily detected at the 4-cell stage and beyond. Culture medium supplemented with 0.5 μg/mL Shh optimized blastocyst rates (58.6 vs. 41.1%; P < 0.05) and the total number of cells per blastocyst (56.4 vs. 45.6 cells; P < 0.05); however, this response was prevented by simultaneous addition of 1 mM cyclopamine (an Shh inhibitor). Moreover, blastocysts that developed in medium containing 0.5 μg/mL Shh had lower apoptotic indices and reduced DNA damage (evaluated by TUNEL and comet assays, respectively). Based on Western-blot analysis, expression of phosphorylated Akt protein in Shh-treated blastocysts was higher than that of the control group (1.22- vs. 0.66-fold, P < 0.05), and less total PARP-1/2 protein was accumulated (0.7-fold, P < 0.05) in treated blastocysts compared to untreated controls. Furthermore, supplementation of Shh (1 μg/mL) also supported development of handmade cloned embryos (50.3 vs. 26.8%; P < 0.05) with reduced apoptotic rates (2.8 vs. 6.3%; P < 0.05). We inferred that the Shh signaling pathway existed in porcine PA embryos and we concluded that Shh supplementation improved the quality and developmental competence of early PA embryos, at least in part, by increasing cell proliferation and reducing apoptosis of the developing embryos.

Comparison of antioxidative/oxidative profiles in blood plasma of cows with and without retained fetal placental membranes - Corrected Proof

2010-07-21

Abstract: Ante- and postpartum antioxidative/oxidative profiles in blood plasma of cows with and without retained placental membranes (RFM) were investigated. Twenty-two healthy pregnant cows were included in the study. Seven animals out of 22 suffered from RFM. Blood samples were obtained at 4, 3, 2, 1 weeks and 5 days antepartum (a.p.), at parturition and 1, 3, and 5 weeks postpartum. The following antioxidative parameters were measured using spectrophotometric methods: total antioxidant activity (TAC), β-carotene, vitamin A, vitamin C, and ceruloplasmin. The oxidative profile was based on the content of intermediates and end products of lipid and protein peroxidative processes which were measured by spectrophotometric and spectrofluorimetric methods. The examined parameters revealed a dynamic profile within the experimental period. The highest antioxidant and oxidant activity was noted at 2 and 1 week a.p. with a drop towards parturition suggesting the presence of oxidative stress during this time period and an apparent appropriate metabolic response of the macroorganism. Except for TAC and vitamin A, the contents of oxidative and antioxidative blood constituents did not differ between cows with and without RFM. A TAC and vitamin A by time interaction indicated higher antepartal concentrations of TAC and vitamin A in cows without RFM than in cows with RFM suggesting a possible role of antioxidative/oxidative imbalances in the aetiology of RFM.

Development of morulae from the oocytes of cultured sheep preantral follicles - Corrected Proof

G. Arunakumari, N. Shanmugasundaram, V.H. Rao — 2010-07-08

Abstract: Sheep preantral follicles (PFs) measuring 250–400 μm in diameter were cultured for six days in serum-free media supplemented differently with growth factors and hormones. Subsequently, oocytes from the cultured follicles were subjected to an additional 24 h of in vitro maturation (IVM) followed by in vitro fertilization (IVF) and embryo culture for 6 days. Five different experiments were conducted. In the first experiment individual concentrations of Insulin-Transferrin-Selenite (ITS), Insulin-like growth factor-I (IGF-I), Transforming growth factor-beta (TGF-β), Insulin (INS), and Growth hormone (GH) that supported the best in vitro development of the PFs were determined. The influence of different combinations of the above hormones and growth factors at their best concentrations as determined in the first experiment was investigated in the second experiment. In the third experiment the best combinations of the growth factors and hormones obtained in the second experiment were additionally supplemented with Thyroxin (T4) and follicle stimulating hormone (FSH) and the influence on in vitro development of the PFs was studied. In the fourth experiment, two methods of culturing PFs—micro drops and agar gel embedding—were compared. In the fifth experiment oocytes from cultured PFs were subjected to IVF and in vitro development of the resulting embryos was followed to the blastocyst stage.Based on the proportion of the PFs exhibiting growth, mean increase in diameter, proportions of PFs developing antrum, ovulations in vitro and oocytes maturing to M-II stage, 1% ITS, 10 ng/mL each of IGF-I, and Insulin and 1 mIU/mL of GH were found to support the best development of sheep PFs. However, the oocytes from PFs cultured in any concentration of TGF-β failed to mature to M-II stage. Similarly, among the combinations studied, IGF-I+GH was found to be the best. In combination with T4 and FSH, IGF-I+GH supported the best development of the PFs. Culture of PFs in micro drops or agar gel supported similarly high development. In vitro fertilization of the oocytes from the cultured sheep PFs resulted in the embryos developing to the morula stage for the first time.

Total RNA isolation from stallion sperm and testis biopsies - Corrected Proof

2010-07-08

Abstract: Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200 × g for 30 min through a 40% EquipureTM silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 μg of total RNA per 100 × 106 sperm. To evaluate RNA quantity and quality, we used a NanoDrop spectrophotometer and Agilent Bioanalyzer. A protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed to determine sperm RNA contamination with genomic DNA or RNA from somatic cells. By these methods, hybridization- and sequencing-quality RNA was isolated from 11 samples of stallion sperm. Stallion testis biopsy with a 14 gauge 22 mm deep biopsy needle yielded ∼12 μg of good quality total RNA, and could serve as an alternative to excision surgery for sample procurement. Compared to RNA isolation from testis, the sperm required advanced processing and RNA quality control. The described methodologies provided a foundation to establish functional genomic studies of stallion fertility.

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus) - Corrected Proof

I. Adachi, S. Kusuda, E. Nagao, Y. Taira, M. Asano, T. Tsubota, O. Doi — 2010-07-08

Abstract: Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.

Differences in semen freezability and intracellular ATP content between the rooster (Gallus gallus domesticus) and the Barbary partridge (Alectoris barbara) - Corrected Proof

2010-07-08

Abstract: This study aimed to compare viability, ATP content, and DNA integrity of rooster (Gallus gallus domesticus) and Barbary partridge (Alectoris barbara) fresh and frozen spermatozoa in order to identify factors possibly related to differences in semen freezability. Ejaculates were obtained from March to May by the abdominal massage method from 3 adult roosters and 12 adult Barbary partridges. Semen was frozen with different cryoprotectants using Lake's diluents as a base medium: 1) glycerol 11%; 2) glycerol 11% and trehalose 70 mmol/L; 3) dimethylacetamide (DMA) 6%; 4) DMA 6% and trehalose 70 mmol/L. Both fresh and frozen semen showed a lower viability and higher intracellular ATP concentrations in the Barbary partridge compared with the rooster (P < 0.05). In the Barbary partridge, semen viability after thawing did not differ among the 4 media used, but glycerol showed positive effects in avoiding a significant loss of ATP after thawing, compared with DMA containing media (P < 0.05). On the other hand, in the rooster a higher viability was recorded when semen was frozen in glycerol containing media compared to DMA (P < 0.0001), while ATP values significantly decreased after thawing (P < 0.05) without showing any differences among the semen frozen in the 4 different media. DNA integrity, as evaluated by the comet assay, was assessed only in frozen semen. In the Barbary partridge, mean scored parameter did not differ significantly among semen frozen in the 4 different media. In the rooster DNA fragmentation was higher in DMA ctr medium compared with the other media and with values found in Barbary partridge semen frozen in the same medium (P < 0.001). In both species, the addition of trehalose did not show any positive effects on viability, ATP levels and DNA integrity after thawing.In conclusion, species-related differences in semen features exist between the rooster and the Barbary partridge and the wide variation observed in ATP levels may account for differences in semen freezabililty between the two species.

The influence of cumulus cells during in vitro fertilization of buffalo (Bubalus bubalis) denuded oocytes that have undergone vitrification - Corrected Proof

2010-07-08

Abstract: The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M–0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control.An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01).In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development.

Intrapulse temporality between pulses of a metabolite of prostaglandin F2α and circulating concentrations of progesterone before, during, and after spontaneous luteolysis in heifers - Corrected Proof

2010-07-08

Abstract: Pulses of the prostaglandin F2α (PGF) metabolite 13,14-dihydro-15-keto-PGF2α (PGFM) and the intrapulse concentrations of progesterone were characterized hourly during the preluteolytic, luteolytic, and postluteolytic periods in seven heifers. The common hour of the end of preluteolysis and the beginning of luteolysis was based on a progressive progesterone decrease when assessed only at the peaks of successive oscillations. The end of the luteolytic period was defined as a decrease in progesterone to 1 ng/mL. Blood samples were taken hourly from 15 d after ovulation until luteal regression as determined by color-Doppler ultrasonography. Between Hours −2 and 2 (Hour 0 = PGFM peak) of the last PGFM pulse of the preluteolytic period, progesterone decreased between Hours −1 and 0, and then returned to the prepulse concentration. Concentration did not change significantly thereafter until a PGFM pulse early in the luteolytic period; progesterone decreased by Hour 0 and transiently rebounded after Hour 0, but not to the prepulse concentration. In the later portion of the luteolytic period, progesterone also decreased between Hours −1 and 0 but did not rebound. After the defined end of luteolysis, progesterone decreased slightly throughout a PGFM pulse. Results demonstrated for the first time that the patterns of progesterone concentrations within a PGFM pulse differ considerably among the preluteolytic, luteolytic, and postluteolytic periods.

Polymyxin B neutralizes bacteria-released endotoxin and improves the quality of boar sperm during liquid storage and cryopreservation - Corrected Proof

2010-07-08

Abstract: Gram negative bacteria are the predominant type detected in boar semen. Since these bacteria release lipopolysaccharide (LPS), and because polymyxin B (PMB) neutralizes LPS activity, the objective was to improve techniques for long-term storage of boar sperm by testing the beneficial effects of PMB. In the present study, LPS bound directly to the head region of sperm, decreased sperm motility, and induced sperm apoptosis. The addition of 100 μg/mL PMB suppressed LPS binding activity and blocked the negative effects of LPS on sperm quality. Additionally, when PMB treatment was combined with penicillin G (PenG), sperm motility was increased after 10 d of liquid storage or in frozen-thawed sperm (P<0.05). When the PMB- and PenG-treated sperm was used for artificial insemination, the conception rate was increased relative to that of artificial insemination with sperm treated by PenG alone in both liquid (62 vs. 81%) and cryopreserved forms (50 vs. 80%, P < 0.05). We concluded that PMB suppressed LPS-induced low sperm motility and apoptosis via the reduction of LPS binding to Toll-like receptors (TLRs). Thus, in order to enhance sperm quality for artificial insemination, a combined treatment with PMB and PenG immediately after ejaculation seemed appropriate to maintain sperm motility and function during both liquid storage and cryopreservation.

Evaluation of newborn canine viability by means of umbilical vein lactate measurement, apgar score and uterine tocodynamometry - Corrected Proof

D. Groppetti, A. Pecile, A.P. Del Carro, K. Copley, M. Minero, F. Cremonesi — 2010-07-08

Abstract: Newborn viability evaluation and early detection of fetal distress could contribute to reducing mortality at birth in canine species. High neonatal mortality rate in dogs is reported subsequent to complicated or uncomplicated whelping. Umbilical vein lactate and tocodynamometry could provide valuable clinical information to the obstetricians so that appropriate medical and surgical treatments or oxygen and warm administration can be properly and timely applied to mother and newborn pup. In humans, the fetal lactate level represents an objective indicator of fetal distress and a valid predictor of babies' survival. Fetal acidosis recognition by umbilical lactate (UL) measurement, APGAR score classification, and uterine activity monitoring during labour, can represent an advanced system in the evaluation of the canine newborn patient. The purpose of this study was to correlate UL levels with canine neonatal morbidity and mortality within 48 h of birth. We evaluated the relationship among neonatal parameters at birth (mucous membrane color, heart and respiratory rate, reflex irritability, mobility, suckling and vocalization, UL, weight, and temperature) with labour characteristics (uterine contractions recorded by the tocodynamometric system of WhelpwiseTM Veterinary Perinatal Specialties®, delivery time, and pup presentation), in view to predict pup viability. We considered also vaginal parturition versus elective and emergency Caesarean section, and uterotonic drugs influence on delivery. Umbilical lactate concentration proved to be useful to predict canine neonatal mortality within 48 h of birth (P < 0.05). We identified 5 mmol/L of vein umbilical lactate concentration as the cut off value, allowing us to distinguish between healthy and distressed pups. Higher values of UL were related with distressed pups, whereas lower values characterized vigorous pups. Lactate concentrations lower than 5 mmol/L and APGAR scores higher than 9, related to mean delivery time of 105 min with effective uterine contractions (10 mm of Hg of strength or more, frequency from 4 to 12 contractions per hour, and 2–5 min in duration), should be considered good prognostic factors in canine labour and neonatology.

Alkaline phosphatase as an indicator of true ejaculation in the rhinoceros - Corrected Proof

2010-07-08

Abstract: The objective was to determine if seminal alkaline phosphatase (ALP) can serve as an indicator of true ejaculation in the rhinoceros. Concentrations of ALP activity were determined in seminal fractions collected from African black rhinos (Diceros bicornis), an African white rhino (Ceratotherium simum), and an Indian rhino (Rhinoceros unicornis) during electroejaculation. In addition, seminal fractions collected during penile massage of a Sumatran rhino (Dicerorhinus sumatrensis) were assessed. Correlations between ALP activity and sperm concentration, fraction pH, and fraction osmolality were evaluated in the Indian rhino and black rhino. Concentrations of ALP activity in rhino ejaculate fractions ranged from < 5 to 11,780 U/L and were positively correlated (P < 0.05) with sperm concentration for both Indian rhino (r = 0.995) and black rhino (r = 0.697), but did not exhibit a strong correlation with either pH or osmolality (P > 0.05). Data were insufficient for establishing meaningful correlation coefficients in the Sumatran rhino and white rhino, but preliminary results were in accordance with findings in the Indian rhino and black rhino. We concluded that ALP was present in rhinoceros semen, likely originated from the epididymides and/or testes, and could serve as a useful tool for assessing the production of ejaculatory versus pre-ejaculatory fluid in the rhinoceros.

The value of the percentage of motile sperm in predicting a significant portion of the fertility variation of frozen-thawed buck semen - Corrected Proof

V. Furstoss, F. Borderes, Y. Forgerit, P. Guillouet, B. Leboeuf — 2010-07-08

Abstract: Prediction of the future fertility of a given ejaculate with a simple laboratory test is still considered a real issue in domestic mammal breeding. This study showed that a subjective assessment of the percentage of motile spermatozoa, measured 120 min after thawing (mob120), can predict a significant part (∼50%) of the variation of the future fertility of buck ejaculates. The predictive model was calculated using a calibration data set composed of 40 ejaculates from four Alpine and six Saanen bucks. A fertility trial using split ejaculates was conducted in order to estimate ejaculate fertility. Taken into account were the herd within breed factor and the year, month, and inseminator factors. On average, one ejaculate was used to inseminate two females per herd in 10 different herds. This calibration set allowed us to choose the mob120 variable among a set of laboratory tests: mitochondrial activity, acrosomal status, membrane integrity, osmotic resistance test assessed by flow cytometry, velocity and motion characteristics assessed by computer-assisted sperm analysis, visually assessed percentage of motile, and motility score measured 5 and 120 min after thawing. For the calibration step, the best model used the logarithm of mob120 and gave a correlation coefficient of 0.71 between the field fertility and the predicted fertility and a standard error of 0.17. We tested this model on 3 different validation data sets adding up to 95 ejaculates that were all different from those of the calibration data set. The correlation coefficients between field fertility and predicted fertility were always significant and the bias corrected standard error ranged from 0.15 to 0.18 on these validation data sets. A Monte Carlo simulation showed that about 20% of the fertility variation remained to be explained.

Seminal plasma affects prostaglandin synthesis in the porcine oviduct - Corrected Proof

2010-07-08

Abstract: Seminal fluids introduced to the female reproductive tract at mating can affect subsequent events, such as ovulation, fertilization, conception, and pregnancy. Bioactive molecules present in seminal plasma can modify the cellular composition, structure, and function of local tissues and of tissues distal to the tract. The oviduct plays a decisive role in reproduction providing a beneficial milieu for gamete maturation, fertilization, and early embryonic development. Therefore we have investigated whether intrauterine infusion of seminal plasma can modulate prostaglandin (PG) synthesis in the porcine oviduct through regulation of gene and protein expression of enzymes of prostaglandin synthesis pathway. Among several enzymes involved in the prostaglandin synthesis pathway tested in the present study PGF2α synthase (PTGFS) and prostaglandin 9-ketoreductase (CBR1), which convert PGE2 to PGF2α, expression were significantly down-regulated in the oviducts on Day 1 after seminal plasma infusion into the uterine horns. The effects of the treatment were transient and by Day 5 levels of PTGFS and CBR1 were comparable in seminal plasma-treated and control animals. Additionally, increased PGE2 to PGF2α and PGFM to PGF2α ratios in the oviductal tissues were indicated. Our results clearly demonstrate that seminal plasma affects prostaglandin synthesis in the porcine oviduct. Altered PTGFS and CBR1 expression in consequence changed PGE2 to PGF2α and PGFM to PGF2α ratios in the porcine oviduct.

Factors affecting pre-ovulatory follicle diameter in the mare: the effect of mare age, season and presence of other ovulatory follicles (multiple ovulation) - Corrected Proof

M.C.G. Davies Morel, J.R. Newcombe, K. Hayward — 2010-07-08

Abstract: The importance of elucidating factors affecting reproductive performance and efficiency is of paramount concern to the equine industry. Oocyte viability is known to be one of the determinants of reproductive success and evidence suggests that it may be linked to follicle size. The aims of this study were, therefore, to ascertain: i) the average diameter and range of pre-ovulatory follicles in Thoroughbred mares; ii) whether this is affected by either mare age, time within the breeding season, or the presence of multiple pre-ovulatory follicles (MO). One thousand, four hundred and ninety two Thoroughbred mares, aged 2–26 years, were examined with ultrasound to ascertain ovulation date to within 24h, and pre-ovulatory follicle(s) (F1) diameter. Mares were divided into groups according to age (7 groups, 2–4 yr, 5–7 yr, 8–10 yr, 11–13 yr, 14–16 yr, 17–19 yr, >19 yr), time within the season (16 half-month groups, from Feb 1st to Sept 30th), and pre-ovulatory follicles (single, {SO} or multiple {MO}). Overall average F1 diameter was 39.95 ± 4.84 mm (range 22–50 mm). Mare age had a significant (P < 0.001) negative effect on F1 diameter (largest F1 38.95 ± 5.61 mm, mares 2–4 yrs; smallest F1 33.30 ± 4.66 mm, mares >19 yrs) as did season (largest F1 44.20 ± 3.95 mm, Feb 1st–14th; smallest F1 33.74 ± 4.87 mm, Aug 15th–31st) and the presence of more than one pre-ovulatory follicle (MO F1 35.45 ± 4.53 mm; SO F1 37.44 ± 4.84 mm). In conclusion older mares, bred towards the end of the breeding season, especially if MO were present, were more likely to ovulate from smaller follicles. If, as suggested, small pre-ovulatory follicle size is associated with low oocyte viability, then this may account, at least in part, for the poor fertility rates characteristic of older MO mares, bred later in the season and so justify increased monitoring and careful reproductive management of such mares.

A clinical approach to determine false positive findings of clinical endometritis by vaginoscopy by the use of uterine bacteriology and cytology in dairy cows - Corrected Proof

S. Westermann, M. Drillich, T.B. Kaufmann, L.V. Madoz, W. Heuwieser — 2010-07-08

Abstract: Clinical endometritis in dairy cows is defined as mucopurulent or purulent vulvar discharge 21 days or more after parturition. The diagnosis of clinical endometritis is commonly based on vaginal examination. Techniques to reduce the proportions of false negative findings have been described. This paper discusses a clinical approach to determine the proportion of false positive findings that might occur by vaginal inspection. The consequences of false positive findings in dairy practice are unnecessary or inadequate treatments. In research, incorrect diagnoses have an impact on the interpretation of studies on the diagnosis and treatment of clinical endometritis. The objective of the present study was to compare intrauterine bacteriology and endometrial cytology in cows diagnosed with clinical endometritis with findings obtained by vaginoscopy. Clinical endometritis was defined as mucopurulent or purulent vulvar discharge. On two commercial dairy farms, cows were examined 21 to 28 d postpartum. Uterine samples (n = 230) were collected from cows with clinical endometritis with the cytobrush technique to determine the proportion of polymorphonuclear neutrophils (PMN) and to culture smears for aerobic bacteria. Two threshold values for the proportion of PMN (5 and 18%) were chosen as possible indicators for an inflamed endometrium. Common uterine pathogens A. pyogenes and E. coli were found in 33.5 and 10.4% of the samples, respectively. With increasing vaginal discharge score, proportion of samples positive for A. pyogenes increased significantly. The proportion of cows exceeding the thresholds for PMN increased with vaginal discharge score and the presence of A. pyogenes.Considering only the presence of aerobic uterine pathogens and a proportion of PMN above the threshold values of 5 and 18% as indicative for endometritis, a proportion of 17.3 and 28.5%, respectively, of diagnoses by vaginoscopy were false positive.

Foreward - Corrected Proof

Fulvio Gandolfi, John Kastelic — 2010-07-08

The Domestic Animal Biomedical Embryology (DABE) committee consists of persons with interests in embryological applications not directly related to reproduction, including transgenesis, cloning, and stem cells. The committee, organized during an informal gathering at the International Embryo Transfer Society (IETS) meeting in 2007, was officially recognized by the IETS Board of Governors in 2008. This committee is open to all the IETS members and meets annually in association with the Society's annual meeting (for more information, visit the IETS home page: http://www.iets.org/hasac.htm).

The use of signalling pathway inhibitors and chromatin modifiers for enhancing pluripotency - Corrected Proof

H. Sumer, J. Liu, P.J. Verma — 2010-07-08

Abstract: Pluripotent embryonic stem cells have been isolated from a limited number of species. The new advances with inducing pluripotency in somatic cells have resulted in the generation of pluripotent stem cells while circumventing the need for embryos. In this review we describe the main signalling pathways involved in maintaining pluripotency and inducing differentiation. Inhibition of the signalling pathways involved in differentiation enhances the derivation and cultivation of pluripotent stem cells. Furthermore, we discuss the use of chromatin modifiers to maintain an open chromatin state which is characteristic of pluripotent stem cells, to facilitate the derivation of pluripotent cell lines.

Cooling and freezing of epididymal sperm in the common hippopotamus (Hippopotamus amphibius) - Corrected Proof

J. Saragusty, C. Walzer, T. Petit, G. Stalder, I. Horowitz, R. Hermes — 2010-07-08

Abstract: Knowledge concerning reproduction in common hippopotamus is scarce and in particular very little is known about male reproductive physiology and sperm cryopreservation. Testes were obtained from nine castrated bulls and sperm extracted from the epididymides of eight of these individuals. Mean ± SEM values of reproductive parameters were: testicular weight (including epididymis and tunicas)—275.9 ± 54.1 g, total sperm motility—88.1 ± 4.2%, total cells extracted—11.0 ± 3.6 × 109, intact acrosome—87.7 ± 1.8%, intact sperm morphology—51.6 ± 4.1%, and, for 3 individuals, hypoosmotic swelling test for membrane integrity—83.3 ± 1.8%. Chilled storage extenders tested were Berliner Cryomedium (BC), Biladyl®, modification of Kenney modified Tyrode's medium (KMT), and Human Sperm Refrigeration Medium (HSRM). Extender had significant effect on post-dilution motility and motility and intact morphology after 4h and 24h at 4°C (P ≤ 0.007 for all). Berliner Cryomedium and HSRM were superior to Biladyl® and KMT. Freezing extenders tested were BC with either 6% dimethyl sulfoxide (Me2SO), or 5%, 7%, or 10% glycerol. Post-thaw motility was < 5% in 3/7 bulls in all extenders. When frozen in BC with 6% Me2SO, one bull had 15% post-thaw motility and 3/7 had 20 to 60%. In glycerol, 3/7 had 15–30% post-thaw motility in 5%, 2/7 in 7%, and 1/7 in 10%. The extender had significant effect on post-chilling motility (P = 0.008), post-thaw morphology (P = 0.016), and motility 30 min after thawing (P = 0.015). Berliner Cryomedium with 6% Me2SO or 7% glycerol were the freezing extenders of choice. Information obtained in this study allows initiation of cryobanking of sperm from the common hippopotamus which is of particular importance for genetically valuable individuals.

Evaluation of mathematical models to describe testicular growth in Blackbelly ram lambs - Corrected Proof

H. Jiménez-Severiano, M.L. Reynoso, S.I. Román-Ponce, V.M. Robledo — 2010-07-02

Abstract: The primary objective was to compare various mathematical models to describe scrotal circumference (SC) and paired testis volume development in Blackbelly ram lambs. The study was conducted in the state of Querétaro, México (20° 43' N, 100° 15' W). Spring-born Blackbelly ram lambs (n = 41) were housed outdoors and fed alfalfa hay and concentrate. Body weight, SC, and testis length, diameter, and volume were recorded every 2 wk from 24 to 172 d of age (June 18 to November 3). The following mathematical functions were used to model SC-age and testis volume-age relationship: Von Bertalanffy, Brody, Gompertz, Logistic, and Richards. The suitability of the models was evaluated based on parameter values and standard errors, residual mean square, the coefficient of determination (R2), and the average prediction error (APE). All models, except for Brody's, had good fit to SC (R2 > 0.98) and testis volume (R2 > 0.95), and produced similar growth curves in the range of ages studied. The logistic model predicted SC at maturity quite well, 33.6 ± 0.6 cm as compared with 33.9 ± 0.5 cm observed in adult animals; all models had APE's smaller than ±7% between 56 and 168 d of age. The Bertalanffy model predicted testis volume at maturity quite well, 513 ± 22 cm3 as compared with 488 ± 20 cm3 calculated for adult animals. The logistic model had a good fit to testis volume during the period of study, but underestimated the volume at maturity by 28%. All models, except for Brody's, had APE's smaller than ±14% between 98 and 168 d of age. The logistic and Bertalanffy models predicted the inflection point for SC at 83 and 59 d of age, and testis volume at 116 and 109 d of age, respectively. In conclusion, all models, except for Brody's, had good fit to actual SC and testis volume data in the range of age evaluated, whereas the logistic and Bertalanffy's models made the best predictions for adult SC and testis volume, respectively.

Economic consequences of reproductive performance in dairy cattle - Corrected Proof

C. Inchaisri, R. Jorritsma, P.L.A.M. Vos, G.C.van der Weijden, H. Hogeveen — 2010-06-28

Abstract: The net economic value of reproductive efficiency in dairy cattle was estimated using a stochastic dynamic simulation model. The objective was to compare the economic consequences of reproductive performance scenarios (“average” and “poor”) of a cow having a good reproductive performance and to explore which reproductive factors have an important impact on economic efficiency. A “good” reproductive performance scenario was defined with 1 ovulation rate (POVUi), 0.7 estrus detection rate (PEst), 0.7 conception rate (PCon), 0.03 incidence rate of postpartum disorders prolonging the ovarian cyclicity (CO), 0.2 incidence rate of postpartum disorders reducing conception (ME), 0.05 embryonic death rate (ED), and voluntary waiting period (VWP) of 9 wks pp (post partum). In the current situation of dairy cows in the Netherlands, an “average” reproductive scenario (0.95 POVUi, 0.5 PEst, 0.5 Pcon, 0.07 CO, 0.27 ME, 0.07 ED and VWP of 12 wks pp) and a “poor” reproductive scenario (0.90 POVUi, 0.3 PEst, 0.3 Pcon, 0.11 CO, 0.33 ME, 0.09 ED and VWP of 15 wks pp) were identified. A sensitivity analysis was performed by comparing changes of single effect of factors in a good and poor scenario with the average scenario. The mean net economic loss (NELi) compared with the good scenario was €34 and €231 per cow per year for the average and poor reproductive performance scenario, respectively. Increasing the calving interval resulted in greater economic loss. The important factors on the cost of reproductive efficiency were the involuntary culling cost and the return of milk production. Variation in PCon, PEst, ME, ED, and VWP had large impacts on economic benefits.

Accuracy of pregnancy specific protein-B test for early pregnancy diagnosis in dairy cattle - Corrected Proof

Juan E. Romano, Jamie E. Larson — 2010-06-28

Abstract: The objective was to evaluate the accuracy of an ELISA for pregnancy specific protein B (PSP-B) for early pregnancy diagnosis in dairy cattle. Blood from lactating (>100 d postpartum) dairy cows (n = 738), was collected on Days 28, 30, and 35 (Day 0 = estrus), analyzed with an ELISA for PSP-B, and the cows designated as pregnant, probable, unlikely, or non-pregnant. Immediately after blood collection, transrectal ultrasonography (TRUS) was done for pregnancy diagnosis, and the results used as a criterion standard test for comparison with PSP-B. At Day 28, 46.3% were diagnosed by TRUS as pregnant. The PSP-B sensitivity was 93.9% on Day 28 and similar on Days 30 and 35. The PSP-B specificity, positive predictive value, negative predictive value, and accuracy were all >94% on Day 28 and similar on Days 30 and 35. However, the accuracy was significantly less compared to TRUS (P < 0.01). The percentage of all samples from cows that were probably pregnant or unlikely to be pregnant was 5.6%. At Days 28, 30, and 35, percentages of uncertain samples were 8.5, 4.8, and 3.3%, respectively (P < 0.01), and Kappa values were 0.92, 0.92, and 0.95. False negative and false positive results were attributed to low concentrations of PSP-B in pregnant animals and to persistence of pregnant concentrations of PSP-B in females with pregnancy loss, respectively. In conclusion, PSP-B ELISA was a sensitive, specific, and accurate test for pregnancy diagnosis (relative to TRUS) at Days 28, 30, and 35 after breeding.

The HSP90AA1 sperm content and the prediction of the boar ejaculate freezability - Corrected Proof

2010-06-28

Abstract: In a previous study we reported that the immunolabelling of GLUT3, HSP90AA1, and Cu/ZnSOD proteins on boar sperm did not show differences between good and poor freezability ejaculates, in terms of a qualitative analysis based on location and reactivity of these proteins at 17 °C and at 240 min post-thaw. Since predicting the ejaculate freezability is considerably important in sperm cryopreservation procedures, the objective of the present study was to quantify the expression of these three proteins in good and poor freezability ejaculates. For this purpose, 10 ejaculates from 9 Piétrain boars were cryopreserved and their sperm quality assessed in the three main steps of the freezing process (17 °C, 5 °C, and 240 min post-thaw). After this assessment, the 10 ejaculates were clustered for freezability on the basis of their sperm progressive motility and membrane integrity at 240 min post-thaw. From the whole ejaculates, only four good and four poor freezability ejaculates displaying the most divergent values were selected for a western blot assay using sperm samples coming from the three mentioned freezing steps. Protein levels through densitometry were significantly different between good and poor freezability ejaculates for Cu/ZnSOD at 240 min post-thaw (P ≤ 0.01) and for HSP90AA1 at 17 °C and 5 °C (P ≤ 0.05). This last finding claims the introduction of tests based on molecular markers in spermatozoa to accurately predict the freezability of ejaculates in order to promote the use of frozen semen on artificial insemination programmes.

Biomarkers of in vivo fertility in sperm and seminal plasma of fertile stallions - Corrected Proof

2010-06-28

Abstract: The global proteome of sperm and seminal plasma of fertile stallions was investigated to determine whether associations with relative in vivo fertility exist. Seven stallions at stud in a commercial breeding station were collected throughout the breeding season and bred to a total of 164 mares to determine conception rates. On three occasions during the breeding season, raw semen was obtained from a regular collection for proteomic analysis using two-dimensional electrophoresis and also assessed for routine semen quality end points. First cycle conception rate was negatively related to ejaculate volume (r = −0.43, P = 0.05) and total IGF1 content (ng) per ejaculate (r = −0.58, P = 0.006), whereas overall pregnancy rate was positively related to sperm concentration (r = 0.56, P = 0.01). The abundance of three proteins known to be involved in carbohydrate metabolism in sperm was positively related to fertility. Furthermore, the abundance of four seminal plasma proteins were identified as being negatively related to fertility; these were identified as kallikrein-1E2 (KLK2), clusterin, and seminal plasma proteins 1 (SP1) and 2 (SP2). Abundance of cysteine-rich secretory protein 3 (CRISP3) was positively related to first cycle conception rate (r = 0.495, P = 0.027) and may provide a good marker of fertility. Based on stepwise regression analysis, clusterin and SP1 in seminal plasma together with sperm citrate synthase were predictive of fertility (r = 0.77, P < 0.0001). This study identified proteins within sperm and seminal plasma that could serve as biomarkers of semen quality and fertility in stallions.

Analysis of selected sperm by density gradient centrifugation might aid in the estimation of in vivo fertility of thawed ram spermatozoa - Corrected Proof

2010-06-28

Abstract: The aim of the present study was to evaluate the effect of selecting a sperm subpopulation by means of a discontinuous density gradient centrifugation (DGC) on the quality of ram thawed semen, and the relationships between sperm parameters assessed in unselected and in selected sperm samples with in vivo fertility after intrauterine artificial insemination (IUI) using unselected sperm samples. Semen samples from twenty males were collected by artificial vagina and cryopreserved following a standard protocol. After thawing, unselected sperm samples were used in an in vivo fertility trial and sperm motility (subjective and objective, assessed by means of CASA) and membrane and acrosomal integrities (microscopy) were evaluated on unselected and selected sperm samples. In addition, plasmalemma integrity (YO-PRO-1/PI), membrane fluidity (Merocyanine 540/YO-PRO-1), mitochondrial activity (Mitotracker Deep Red/YO-PRO-1), and DNA fragmentation index (%DFI) assessed by Sperm Chromatin Structure Assay (SCSA®) were evaluated by flow cytometry before and after sperm processing using DGC. Results showed that DGC improved all sperm parameters significantly, except the %DFI, which increased after the selection procedure. No relationships were found between sperm parameters evaluated in unselected sperm samples and in vivo fertility. However, we found a positive correlation between spermatozoa with high membrane fluidity within the viable sperm population (VIABMerocyanine+) evaluated in selected sperm samples and in vivo fertility (r = 0.370, P = 0.019). In conclusion, our results suggest that selected spermatozoa represent a sperm subpopulation different to the unselected one that could be related with the in vivo fertility.

Effect of timing of second prostaglandin F2α administration in a 5-day, progesterone-based CO-Synch protocol on AI pregnancy rates in beef cows - Corrected Proof

W.D. Whittier, R.K. Kasimanickam, J.F. Currin, H.H. Schramm, M. Vlcek — 2010-06-28

Abstract: The objective was to compare the timed AI pregnancy rate of Angus-cross beef cows synchronized with a 5-d CO-Synch + CIDR (a progesterone-releasing intravaginal insert) protocol and given two doses of PGF2α (PGF), with the first dose in conjunction with CIDR withdrawal on Day 5, and the second dose given either early or late relative to the first dose. All cows (N = 1782) at 16 locations received 100 μg of GnRH + CIDR on Day 0. Cows received 25 mg of PGF concurrent with removal of the CIDR on Day 5, and were randomly allocated within locations to receive a second PGF either early (N = 881; from 0.5 to 3.9 h) or late (N = 901; from 4.5 to 8.15 h) relative to the first PGF treatment. On Day 8 (72 h after CIDR removal), all cows were inseminated and concurrently given 100 μg of GnRH. Cows were fitted with a pressure-sensitive mount detection device (Kamar) at CIDR removal. Cows were observed twice daily through Day 7 and at the time of AI on Day 8 for estrus and Kamar status (estrus – red, partial and lost Kamar versus no estrus – white Kamar) was recorded. Accounting for location, season, AI sire, cow observed in estrus or not at or before timed AI, and treatment by cows observed in estrus interaction, timed AI pregnancy rates were greater for the late (6.45 ± 0.03 h) than the early (2.25 ± 0.05 h) interval, 57.2 vs. 52.7%, respectively (P < 0.05). In conclusion, cows that received the second PGF late after the first PGF on the day of CIDR removal in a 5 d CO-Synch + CIDR synchronization protocol had significantly higher timed AI pregnancy rates than those receiving the second PGF early after the first PGF.

Evaluation of fresh and frozen-thawed fowl semen by flow cytometry - Corrected Proof

Agnieszka Partyka, Wojciech Niżański, Ewa Łukaszewicz — 2010-06-28

Abstract: The aim of this study was to assess the spermatozoal viability, acrosome integrity, mitochondrial activity, and DNA status in the frozen-thawed fowl semen with the use of flow cytometry. The experiment was carried out on 10 sexually adult roosters of meat type line Flex. The semen was collected three times a week by dorso-abdominal massage method, then pooled and subjected to cryopreservation using “pellet” method and Dimethylacetamide (DMA) as a cryoprotectant. For cytometric analysis the fresh and frozen-thawed semen was extended with EK diluent to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI). Acrosomal damages were evaluated using phycoerythrin-conjugated lectin PNA from Arachis hypogaea. The percentage of live spermatozoa with functional mitochondria was estimated using Rhodamine 123 (R123) and PI. The spermatozoal DNA integrity was measured by sperm chromatin structure assay (SCSA). The freezing-thawing process decreased the viability, mitochondrial activity in the chicken sperm and increased the percentage of dead cells with ruptured and intact acrosomes, and also the percentage of spermatozoa with fragmented DNA. In conclusion, the present study indicates that fluorescent staining and flow cytometry may be useful for assessment of the changes of fowl semen quality caused by cryopreservation process. This technique allows precise examination of spermatozoa functional characteristic in a very short time.

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age - Corrected Proof

2010-06-28

Abstract: The objective was to investigate the effects of developmental stage (fully-expanded or expanding blastocysts) and/or age (harvested on Days 7 or 8) on post-vitrification in vitro survival of bovine blastocysts derived from intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Post-warming survival (re-expansion of blastocoele within 24 h) of ICSI-derived fully-expanded blastocysts (80%) was similar to that of their IVF-derived counterparts (88%). However, the ability of ICSI-derived expanding blastocysts to survive vitrification procedures (61%) was lower than that of IVF-derived blastocysts (85%; P < 0.05), although the ICSI- and IVF-derived fresh blastocysts were of similar quality. The age of the blastocysts before vitrification did not affect cryotolerance for either ICSI-derived (73 and 59% for Days 7 and 8 embryos, respectively) or IVF-derived blastocysts (86% for both Days 7 and 8 embryos). At 24 h of post-warming culture, ICSI-derived blastocysts surviving vitrification contained a higher proportion of dead cells than their IVF-derived counterparts (5–13% vs. 2–4%; P < 0.05), but these proportions were not different from those of fresh control embryos. There was an adverse effect of vitrification on the ability of blastocysts to hatch within 72 h of culture only in IVF-derived Day 8 blastocysts (41 and 70% in vitrified and fresh control groups, respectively). In conclusion, the proportion of blastocysts that survived vitrification procedures was similar for ICSI- and IVF-derived bovine blastocysts if the former were cultured to the fully-expanded stage prior to vitrification, with no significant difference between embryos harvested on Day 7 versus Day 8.

Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm - Corrected Proof

E. Breininger, P.D. Cetica, M.T. Beconi — 2010-06-28

Abstract: The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27–29%) and acrosome-reacted sperm (23–26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.

Influence of the thawing rate on the cryopreservation of semen from collared peccaries (Tayassu tajacu) using Tris-based extenders - Corrected Proof

2010-06-28

Abstract: The objective was to evaluate the influence of the thawing rate on the quality of frozen-thawed (cryopreserved in Tris-based extenders) semen obtained from collared peccaries (Tayassu tajacu). Semen from 13 sexually mature collared peccaries males were collected by electroejaculation, and evaluated for motility, vigor, sperm viability, membrane integrity, and sperm morphology. Semen was divided in two equal portions: the first was diluted in Tris-fructose and the other in Tris-glucose, with egg yolk (20%) and glycerol (3%) added to each portion. Extended semen was frozen in liquid nitrogen and thawed using two thawing protocols (37 °C for 1 min or 55 °C for 7 s, followed by an additional 30 s at 37 °C). There were no significant differences between the two extenders after extension, chilling, or glycerol addition. After thawing at 37 °C, there were 37.9 ± 4.2% and 28.5 ± 5.1% motile spermatozoa for samples extended in Tris-fructose and Tris-glucose, respectively, with 33.8 ± 3.7% and 28.2 ± 3.5% motile spermatozoa after thawing at 55 °C (no significant differences). Furthermore, there were no significant interactions between extenders and thawing protocols for any semen end point. In conclusion, semen from collared peccaries was successfully cryopreserved in Tris-based extenders and thawed with two protocols (37 °C for 1 min or 55 °C for 7 s).

Identification of bovine CD52-like molecule by monoclonal antibody IVA-543: distribution of CD52-like molecule in the bull genital tract - Corrected Proof

2010-06-28

Abstract: The bovine maturation-associated sperm membrane antigen CD52-like molecule has been analysed using a mouse anti-sperm monoclonal antibody developed against bull spermatozoa. The antigen recognised by monoclonal antibody IVA-543 was detected on blood mononuclear cells (including lymphocytes and monocytes) and on a minor population of polymorphonuclear leukocytes. The bovine CD52-like molecule is secreted by the epididymal epithelium and then it is inserted into the sperm membrane during the epididymal transport in the distal part of epididymis. The CD52-like molecule was absent from spermatozoa derived from testes, and the highest proportion of IVA-543-reactive sperm was observed in the cauda epididymis (91.6%).This study has shown that the new molecule identified on bovine cells has properties analogous to those previously described for CD52 molecules in man, mouse, rat, monkey, and dog.

Changes in the histomorphology of the canine cervix through the oestrous cycle - Corrected Proof

S. Goericke-Pesch, B. Schmidt, K. Failing, A. Wehrend — 2010-06-28

Abstract: The importance of canine reproduction is steadily increasing and little is known about the canine cervix so far. The aim of this study was to describe the histomorphology of the canine cervix and to determine its correlation to the stage of oestrous cycle and to circulating concentrations of progesterone (P4) and estradiol-17beta (E2), respectively. Ovariohysterectomy (OHE) was performed in 33 non-pregnant, healthy, intact bitches in defined stages of oestrous cycle (proestrus N = 5, estrus N = 7, early diestrus N = 5, late diestrus N = 11, anestrus N = 5). The entire cervix was collected for histological evaluation of the epithelial layer (number of layers, thickness), the stroma (number of layers, thickness, density, structure, and distribution of elastic fibres) and the average area and density of cervical glands as well as blood vessels. These parameters were evaluated in all the three parts of the cervix (cranial, middle, and caudal or vaginal part). The cervix showed the typical structure with a superficial epithelium, a lamina propria with cervical glands and vascular structures and a tunica muscularis below. Folds in the superficial epithelium were only observed in 54% females (N = 18). Epithelial thickness (P < 0.0001), number of glands (P < 0.05), mean area of glands (P < 0.0001), mean area of venous vessels (P < 0.0001), number of arterial vessels (P = 0.02), number of mast cells and number of eosinophilic granulocytes per mm2 (P < 0.01) were significantly influenced by the stage of cycle. The following factors were significantly influenced by the localisation: number of epithelial layers (P < 0.0001), thickness of stroma (P < 0.0001) and mean number of glands (P < 0.01). Only mean area of venous vessels was significantly influenced by the stage of cycle and the localisation (P < 0.01). Besides, P4 was significantly correlated to number of glands per mm2 (P < 0.0001), mean area of venous vessels (P < 000.1) and number of mast cells (P < 0.01).This study provided detailed information about the histomorphological structure of the cervix in non-pregnant bitches and showed that the cervix undergoes cyclic changes during the canine oestrous cycle, in particular in association with circulating progesterone concentration.

Umbilical artery doppler sonography in Saanen goat fetuses during singleton and multiple pregnancies - Corrected Proof

G. Serin, Ö. Gökdal, T. Tarımcılar, O. Atay — 2010-06-28

Abstract: The objective of this study was to evaluate the blood flow from the umbilical artery (UA) in healthy pregnant goats. Doppler sonography examinations were performed every two weeks in Saanen goats with a singleton (n = 5) or multiple (n = 4) pregnancy from 40 to 145 days of gestation. Fetal heart rates (FHR), pulsatility index (PI), and resistance index (RI) were recorded from the mid-cord site of the free-floating umbilical cord. FHR decreased gradually as the pregnancy progressed and significantly decreased during the last two examinations of all fetuses (P < 0.05). The mean PI level was dramatically different (P < 0.05) until 85 days of gestation, after which it reached a plateau level until parturition. Similar to PI, RI decreased by 85 days of gestation (P < 0.05), and decreased again by 130s gestation. No reverse or absent end-diastolic flow were observed in fetuses during any examinations. When comparing singleton and multiple pregnancies, there were no significant differences in UA pulsatility or resistance in fetuses seen. The middle of the second trimester was observed to be a threshold stage for indices in the pattern of caprine pregnancy.In conclusion, this work provides additional values that might be useful when evaluating singleton and multiple pregnancies, and may be evaluated in further studies regarding fetal monitoring.

Differences in testosterone, androstenone, and skatole levels in plasma and fat between pubertal purebred Duroc and Landrace boars in response to human chorionic gonadotrophin stimulation - Corrected Proof

I.C. Oskam, S. Lervik, H. Tajet, E. Dahl, E. Ropstad, Ø. Andresen — 2010-06-28

Abstract: The concentrations of the boar taint compounds androstenone and skatole in plasma and fat, together with those of testosterone in plasma, were investigated in pubertal purebred Duroc and Landrace boars following stimulation with human chorionic gonadotrophin (hCG). Higher initial levels of androstenone and testosterone were found in Duroc than Landrace boars. Duroc boars, which were approximately ten days older than the Landrace boars, also showed a more advanced stage of spermatogenesis than Landrace boars. While Landrace boars had the highest skatole levels. Following stimulation with hCG the relative increases in testosterone, androstenone, and skatole concentrations were highest in Landrace boars. The level of androstenone in fat three days after hCG stimulation exceeded 1 μg/g fat in all stimulated boars. The decreases in plasma levels of androstenone and testosterone on Days 2 and 3 after hCG stimulation were more pronounced in Landrace than Duroc boars. However, unlike the plasma androstenone and testosterone levels, the plasma concentrations of skatole did not decrease on Days 2 and 3 following stimulation, but remained elevated on Day 3. These results indicate that the lower levels of testicular steroids in Landrace boars compared with Duroc boars was not due to a lower production capacity, but more likely to a faster dissapearance of steroids in Landrace boars. In the present study, age, live weight, and testicular development did not significantly contribute to the variation in fat androstenone. The present data and previous reports on candidate genes related to androstenone biosynthesis and metabolism suggests that future selection against factors associated with boar taint remains a possible solution for the problem of boar taint in the swine industry.

Assessing reproductive patterns and disorders in free-ranging dogs in Jodhpur, India to optimize a population control program - Corrected Proof

2010-06-28

Abstract: The objectives were to test the hypothesis that estrus and pregnancy are seasonal in free-ranging female dogs (>3 mo old) in Jodhpur, India, and to determine litter size, and the prevalence of fetal resorption in this population. The prevalence of estrus and pregnancy was determined in 5400 free-ranging bitches (trapped and released) at the time of ovariohysterectomy. In a separate study, the uteri and ovaries of 246 free-ranging bitches were examined to determine litter size and fetal resorption. The bitches exhibited seasonal estrus and pregnancy (P < 0.00001), with a higher percentage of bitches in estrus or pregnant during the late monsoon season (September to November) compared to the other three seasons. The mean litter size based on embryo/fetal counts was 4.6 (95% CI = 4.0–5.3; n = 40) and based upon placental site counts was 4.4 (95% CI = 3.9–4.8; n = 105). Prevalence of fetal resorption was 32.6% (95% CI = 20.5–47.5; n = 43) with a mean of 2.8 resorptions per litter in those with at least one resorption (95% CI = 1.8–3.8; n = 14). This was the first study to estimate previous litter size of non-pregnant, free-ranging dogs based upon placental sites. Litter size data from this study will be used in a population demographic model to predict the long-term impact of animal birth control (ABC) on the free-ranging dog population in Jodhpur. Increasing the efforts to surgically sterilize bitches prior to the time of year of peak pregnancy or whelping will help maximize the impact of an ABC program on the Jodhpur free-ranging dog population.

Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species - Corrected Proof

S. Ledda, L. Bogliolo, D. Bebbere, F. Ariu, S. Pirino — 2010-06-28

Abstract: Primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Murine PGC can be easily identified using markers as alkaline phosphatase content or the expression of pluripotent markers such as Pou5f1, Nanog, Sox2, Kit, SSEA1, and SSEA4. These tools allowed us to clarify certain aspects of the complex interactions of somatic and germinal cells in the establishment of the germ cell lineage, its segregation from the neighbouring somatic tissue, and the guidance mechanisms during migration that direct most of the germ cells into the genital ridges. Few data are available from other domestic animals and here we reported our preliminary studies on the isolation, characterization, and in vitro culture of sheep PGCs. Sheep PGCs can be identified with the markers previously used in mouse, but, in some cases, these markers are not coherently expressed in the same cell depending on the grade of differentiation and on technical problems related to commercial antibodies used. Pluripotency of PGCs in culture (EGCs) from domestic animals also needs further evaluation even though the derivation of embryonic pluripotent cell lines from large mammals may be an advantage as they are more physiologically similar to the human and perhaps more relevant for clinical translation studies. Comprehensive epigenetic reprogramming of the genome in early germ cells, and derived EGCs including extensive erasure of epigenetic modifications, may be relevant for gaining insight into events that lead to reprogramming and establishment of totipotency. EGCs can differentiate in vitro in a various range of tissues, form embryonic bodies, but in many cases failed to generate tumours when transplanted into immunodeficient mice and are not able to generate germline chimeric animals after their transfer. Such incomplete information clearly indicates the urge to improve the studies on derivation of stem cells in farm animals and shows the need for a multidisciplinary investigation in order to create farm animal models to set up suitable ethical and technical systems for cell regenerative therapies in humans.

Heritability of semen charcteristics in dogs - Corrected Proof

G.C.W. England, L. Phillips, S.L. Freeman — 2010-06-28

Abstract: Retrospective analysis was performed on semen collected from 24 dogs (parents: 14 Labrador retrievers and 10 Golden retrievers) aged between 16 and 28 months of age. The dogs were part of a large breeding programme but lived in the homes of volunteer families. The semen was subjected to a standardised examination procedure including assessment of: percentage normal motility, sperm concentration, total sperm output, percentage of live normal sperm, and total number of live normal sperm. Semen was subsequently collected from one son of each of the parents when the offspring were aged between 16 and 28 mo (offspring: 14 Labrador retrievers and 10 Golden retrievers), and was subjected to the same examination procedures conducted by the same technician. Examination of breeding records demonstrated that each of the 48 dogs achieved at least one pregnancy within a period of 3 months before to 3 months after the semen collection.There was a weak correlation between parents and offspring for each of the 5 semen parameters, although none of these were statistically significant. Narrow sense heritability measures were low for all parameters except for the heritability of high sperm motility (rN = 0.57) and the heritability of low total sperm output (rN = 0.57).It is plausible that breeding selection procedures may be useful in dog breeding programmes in an attempt to improve semen quality, although any impact upon fertility is yet to be proven.

Erratum to “B-Mode ultrasonographic evaluation of the testis in relation to serum testosterone concentration in male Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis) during the breeding season” Theriogenology 73 (2010) 383–391 - Corrected Proof

2010-06-28

The above paper was published with errors in on page 385. In the original published paper, left and right testis lengths (cm) of AB and TT were transposed. Therefore, TT should have been 5.60 and 5.80 for the left and right testis, respectively, whereas AB should have been 27.00 and 29.00. The complete and correct is reprinted on the next page. We regret any inconvenience that this error has caused.

Oocyte secreted factors improve embryo developmental competence of cocs from small follicles in prepubertal goats - Corrected Proof

2010-06-09

Abstract: Oocytes secrete soluble paracrine factors called Oocyte Secreted Factors (OSFs) which regulate the cumulus cell phenotype. Follicle populations in ovaries from prepubertal females have smaller diameters than their adult counterparts. Oocytes from small follicles are less competent than those from large follicles. The aim of this study was to investigate, in prepubertal goats, the effect of OSFs secreted by denuded oocytes (DOs) from small (<3 mm) or large (≥3 mm) follicles during IVM on embryo development and the blastocyst quality of cumulus-oocyte complexes (COCs) from small follicles and to determine if GDF9 participates in this process. Treatment groups were: (A) COCs non selected by their follicle size (control group); (B) cumulus oocytes complexes from small follicles (SFCOCs), (C) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from small follicles (SFCOCs + SFDOs), and (D) cumulus oocytes complexes from small follicles co-cultured with denuded oocytes from large follicles (SFCOCs + LFDOs). The effect of the addition of kinase inhibitor SB-431542, which antagonizes GDF9, was tested in A, C, and D treatment groups. Co-cultured SFCOCs with SFDOs or LFDOs significantly augmented the blastocyst rate in comparison to SFCOCs alone (15.77%, 17.39% vs. 10.31%, respectively). Blastocysts from SFCOCs + LFDOs group showed higher rates of tetraploid nuclei than blastocysts from SFCOCs and the control group (14.43% vs. 5.45% and 5.24%, respectively; P < 0.05). However, we did not observe differences in the hatching rate, mean cell number or embryo cryotolerance (P > 0.05) between the four treatment groups. The addition of SB-431542 during IVM did not have any effect on blastocyst rate (P > 0.05). In conclusion, in prepubertal goats, COCs with a low embryo developmental competence as a consequence of follicle size can be improved by coculturing them with denuded oocytes from both small and large follicles. GDF9 does not seem play a role in this improvement.

Effects of exogenous melatonin on in vivo embryo viability and oocyte competence of undernourished ewes after weaning during the seasonal anestrus - Corrected Proof

M.I. Vázquez, J.A. Abecia, F. Forcada, A. Casao — 2010-06-07

Abstract: This study investigated the effects of exogenous melatonin on embryo viability and oocyte competence in post-partum undernourished ewes during the seasonal anestrus. At parturition (mid-Feb), 36 adult Rasa Aragonesa ewes were assigned to one of two groups: treated (+MEL) or not treated (−MEL) with a subcutaneous implant of melatonin (Melovine®, CEVA) on the day of lambing. After 45 d of suckling, lambs were weaned, ewes were synchronized using intravaginal pessaries, and fed to provide 1.5× (Control, C) or 0.5× (Low, L) times daily maintenance requirements. Thus, ewes were divided into four groups: C−MEL, C+MEL, L−MEL, and L+MEL. At estrus (Day=0), ewes were mated. At Day 5 after estrus, embryos were recovered by mid-ventral laparotomy and classified based on their developmental stage and morphology. After embryo collection, ovaries were recovered and oocytes were classified and selected for use in in vitro fertilization (IVF). Neither diet nor melatonin treatment had a significant effect on ovulation rate and on the number of ova recovered per ewe. Melatonin treatment significantly improved the number of fertilized embryos/corpus luteum (CL) (−MEL: 0.35 ± 0.1, +MEL: 0.62 ± 0.1; P = 0.08), number of viable embryos/CL (−MEL: 0.23 ± 0.1, +MEL: 0.62 ± 0.1; P < 0.01), viability rate (−MEL: 46.6%, +MEL: 83.9%; P < 0.05), and pregnancy rate (−MEL: 26.3%, +MEL: 76.5%; P < 0.05). In particular, exogenous melatonin improved embryo viability in undernourished ewes (L−MEL: 40%, L+MEL: 100%, P < 0.01). Neither nutrition nor exogenous melatonin treatments significantly influenced the competence of oocytes during IVF. Treatment groups did not differ significantly in the number of healthy oocytes used for IVF, number of cleaved embryos, or number of blastocysts and, consequently, the groups had similar cleavage and blastocyst rates. In conclusion, melatonin treatments improved ovine embryo viability during anestrus, particularly in undernourished post-partum ewes, although the effects of melatonin did not appear to be mediated at the oocyte competence level.

Maedi-Visna virus was detected in association with virally exposed IVF-produced early ewes embryos - Corrected Proof

2010-06-07

Abstract: The objective of this study was to determine whether MVV can be transmitted by ovine embryos produced in vitro and whether the zona pellucida (ZP) provides any protection against MVV infection.Zona pellucida (ZP)-intact and ZP-free embryos, produced in vitro, at the 8–16 cell stage, were cocultured for 72h in an insert over an ovine oviduct epithelial cell (OOEC)-goat synovial membrane (GSM) cell monolayer that had been previously infected with MVV (K1514 strain). The embryos were then washed and transferred to either direct contact or an insert over a fresh GSM cell monolayer for 6 h. The presence of MVV was detected using RT-PCR on the ten washing fluids and by the observation of typical cytopathic effects (CPE) in the GSM cell monolayer, which was cultured for 6 weeks.This experiment was repeated 4 times with the same results: MVV viral RNA was detected using RT-PCR in the first three washing media, while subsequent baths were always negative. Specific cytopathic effects of MVV infection and MVV-proviral DNA were detected in GSM cells that were used as a viral indicator and cocultured in direct contact or as an insert with MVV-exposed ZP-free embryos. However, no signs of MVV infection were detected in cells that were cocultured with exposed ZP-intact or non-exposed embryos.This study clearly demonstrates that (i) in vitro, ZP-free, early ovine embryos, which had been exposed to 103 TCID50/m MVV in vitro, are capable of transmitting the virus to susceptible GSM target cells, and that (ii) the IETS recommendations for handling in vivo produced bovine embryos (use of ZP-intact embryos without adherent material and performing ten washes) are effective for the elimination of in vitro MVV infection from in vitro produced ovine embryos. The absence of interaction between ZP-intact embryos and MVV suggests that the in vitro produced embryo zona pellucida provides an effective protective barrier.

Effects of long-term treatment with the GnrH agonist deslorelin (Suprelorin®) on sexual function in boars - Corrected Proof

Johannes Kauffold, Hartmut Rohrmann, Julia Boehm, Axel Wehrend — 2010-06-07

Abstract: Immunization against GnRH has been proven effective for boar taint removal, and long-term treatment with GnRH analogues has been shown to suppress GnRH dependent reproductive processes in several species. This study was conducted to treat boars (n = 5) with Suprelorin®, i.e., an implant that contains 4.7 mg of the long-acting GnRH analogue deslorelin, and to test the effects on sexual function. Insertion of the implant occurred at the age of 5 weeks and animals were observed until market age at 26–27 weeks. Surgically castrated (n = 4) and intact boars (n = 3) served as controls. Testes growth was markedly reduced and steroidogenesis (testosterone, estrone, estrone sulphate, estradiol 17β) as well as spermatogenesis suppressed in 4 of 5 GnRH treated boars, respectively. The remaining fifth boar resumed testes growth after week 17 of age and had high hormone concentrations when tested at weeks 26 and 27. Restoration of spermatogenesis was observed at 34 weeks of age. There were no effects of treatment on general health, nor were there local inflammatory reactions. Results indicate that suppression of sexual functions in boars due to long-term treatment with the GnRH agonist deslorelin through an implant such as Suprelorin® is possible and can last for several months up to market age; thus it has potential as an alternative to other methods used for boar taint removal. Because the maximum duration of suppression seems to vary between boars, further studies are necessary to refine the treatment.

Effect of liquid storage on sorted boar spermatozoa - Corrected Proof

2010-06-07

Abstract: A routine use of boar sexed semen is far from being a reality due to many limiting factors among which is the long sorting time necessary to obtain the adequate number of sexed spermatozoa for artificial insemination and the high susceptibility to damages induced by cryopreservation.The aim of this study was to evaluate the modification induced by 24–26 h storage on sorted boar spermatozoa on the basis of their viability, acrosome status, Hsp70 presence, and in vitro fertilizing ability. The percentage of viable cells, according to SYBR green/PI staining, was negatively affected (P < 0.05) by sorting procedure. Moreover, liquid storage significantly (P < 0.05) reduced membrane integrity of sorted spermatozoa as compared to all the other groups. Neither sorting nor storage influenced the percentage of live cells with reacted acrosome, according to FITC-PNA/PI staining. Sorted samples, after 24–26 h storage, were characterized by an increase (P < 0.05) of sperm cells negative for Hsp70, as observed by immunofluorescence, and by a decrease (P < 0.05) in Hsp70 content, as evidenced by western blot. While sorting procedure did not adversely affect both penetration rate and total efficiency of fertilization, these parameters were negatively (P < 0.05) influenced by storage after sorting. In order to minimize damages that compromise fertility and function of sex-sorted boar spermatozoa, the mechanisms by which sorting and liquid storage cause these injures require further study.

CpG methylation modulates tissue-specific expression of a transgene in chickens - Corrected Proof

2010-06-07

Abstract: The use of genetically modified germ cells is an ideal system to induce transgenesis in birds; the primordial germ cell (PGC) is the most promising candidate for this system. In the present study, we confirmed the practical application of this system using lentivirus-transduced chicken gonadal PGCs (gPGCs). Embryonic gonads were collected from 5.5-d old Korean Oge chickens (black feathers). The gPGC population was enriched (magnetic-activated cell sorting technique) and then they were transduced with a lentiviral vector expressing enhanced green fluorescent protein (eGFP), under the control of the Rous sarcoma virus (RSV) promoter. Subsequently, the eGFP-transduced PGCs were transplanted into blood vessels of 2.5-d-old embryonic White Leghorn (white feathers). Among 21 germline chimeric chickens, one male produced transgenic offspring (G1 generation), as demonstrated by testcross and genetic analysis. A homozygous line was produced and maintained through the G3 generation. Based on serum biochemistry, there were no significant physiological differences between G3 homozygotes and non-transgenic chickens. However, since eGFP transgene expression in G3 chickens varied among tissues, it was further characterized by Western blotting and ELISA. Furthermore, there were indications that DNA methylation may have affected tissue-specific expression of transgenes in chickens. In conclusion, the PGC-mediated approach used may be an efficient tool for avian transgenesis, and transgenic chickens could provide a useful model for investigating regulation of gene expression.

Development of vitrified bovine secondary and primordial follicles in xenografts - Corrected Proof

2010-06-07

Abstract: The objective was to evaluate the effect of various vitrification conditions on the morphology of bovine secondary and primordial follicles, and to use xenografting to confirm their developmental ability. Secondary follicles were placed in vitrification solution containing 15% (v:v) ethylene glycol (EG), 15% (v:v) dimethyl sulfoxide (DMSO), 20% (v:v) fetal calf serum (FCS), and 0, 0.25, or 0.5 M sucrose at room temperature for 1 or 30 min, or at 4 °C for 30 min before being plunged into liquid nitrogen (LN2). Ovarian tissues with primordial follicles were equilibrated in a solution containing 7.5% EG, 7.5% DMSO, and 20% FCS for 5 or 15 min, and then treated with a vitrification solution (15% EG, 15% DMSO, and 20% FCS) containing 0 or 0.5 M sucrose at room temperature for 1 min, and then plunged into LN2. One week later, follicles and tissues were warmed, and morphology assessed histologically. Secondary follicles vitrified in sucrose-free solution had more oocytes with shrinkage of the nucleus and abnormal cytoplasm relative to those vitrified in sucrose-containing solution. When primordial follicles were equilibrated for 5 min and vitrified in sucrose-free solution, the percentage of morphologically normal primordial follicles was higher than in the other groups (P < 0.05). After 4 wk and 6 mo of xenografting of vitrified-warmed secondary and primordial follicles, respectively, in SCID mice, follicles developed to the antral stage and oocytes grew. In conclusion, bovine secondary follicles were successfully cryopreserved in sucrose-containing vitrification solutions and maintained their ability to develop to the antral stage and grow oocytes, whereas primordial follicles vitrified in sucrose-free solution maintained their morphology and developed to the antral stage, with oocyte growth.

Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezing - Corrected Proof

2010-06-07

Abstract: The cryopreservation of oocytes is valuable for the preservation of women's fertility and might also be an interesting tool to preserve animal genetic biodiversity but it is not often used because of the very poor fertility recovered after thawing, especially in rabbit species. The objective of our study was to evaluate the effect of slow-freezing and vitrification on the structural integrity of ovulated rabbit oocytes, their ATP contents, and their developmental competence. Results show that, whatever the method is used, cryopreservation has a dramatic effect on the metabolic integrity, the structural integrity, and the developmental ability of the oocytes. Vitrification and slow freezing both impair the rabbit oocytes viability after thawing but the processes act differently. Further studies are needed to improve the cryopreservation techniques in rabbit species. Moreover, we underlined that morphology and maintenance of the structural integrity of the oocytes are not suitable enough to assess the potential for further development of cryopreserved MII oocytes. The assessment of ATP metabolism allows efficient evaluation of the viability of the frozen or vitrified oocytes. It should be used in addition to parthenogenesis to better assess the potential for further development.

A description of the findings from bull breeding soundness evaluations and their association with pregnancy outcomes in a study of western Canadian beef herds - Corrected Proof

Cheryl L. Waldner, Richard I. Kennedy, Colin W. Palmer — 2010-06-07

Abstract: The primary objectives were to describe beef bulls considered for use and those reported as used in 205 beef herds in western Canada, and to determine whether factors typically assessed during breeding soundness evaluations were associated with reproductive success. More than 100 veterinary clinics reported 2990 breeding soundness evaluations for bulls considered for natural service in client's herds. Differences among clinics explained 5.2% of the variation in scrotal circumference (SC) and 6.9% of the variation in percentage of morphologically normal sperm of all bulls considered for use (after accounting for age, breed, body condition, significant physical abnormalities, month, and year). The percentage of morphologically normal sperm was lower in bulls with an SC ≤34 versus >34 cm (P < 0.006). This study included data from 1384 and 1370 bulls used for breeding in 2001 and 2002, respectively. Most (80%) of the bulls used were Simmental, Black Angus, Charolais, Red Angus, or Hereford, and 80% were ≤4 y of age. Before the breeding season, a veterinarian evaluated 89.5% of all bulls used in these herds. Of the bulls subjected to a breeding soundness evaluation and subsequently used, 93.1% were satisfactory. In 2001 and 2002, injuries were reported in 2.5 and 2.1% of bulls and in 16.6 and 11.4% of herds, and necrobacillosis of the foot was reported in 2.5 and 1.2% of bulls and 11.2 and 6.5% of the herds. The average number of cows exposed to each bull was 26 (both years). Cows exposed to bulls with a smaller SC were less likely to be diagnosed pregnant (P < 0.047) and had a longer median interval from first bull exposure to calving (P < 0.016) than bulls with a larger SC. In conclusion, our findings emphasized the value of breeding soundness evaluations, including measurements of SC, in fertility management of beef cattle.

Vaginal deposition of frozen-thawed semen in Norwegian Dairy goats: Comparison of single and double insemination with equal total number of spermatozoa - Corrected Proof

A.B. Nordstoga, L. Söderquist, T. Ådnøy, W. Farstad, H. Paulenz — 2010-06-07

Abstract: In a field trial, a total of 472 Norwegian Dairy goats showing natural estrus were artificially inseminated with frozen-thawed semen. The farmers themselves performed vaginal deposition of 400 × 106 spermatozoa; one half of the does received two straws (200 × 106 spermatozoa/straw) at the same time (single AI), while the other half received two straws (200 × 106 spermatozoa/straw) 12 h apart (double AI). The commercially available extender Andromed® was used for dilution. The does were housed at 15 different farms, and on average 31 does were inseminated per farm. Non return rates (NRR) and kidding rates after single insemination were 64.3% and 58.3%, respectively. Double inseminations resulted in a NRR of 62% and a kidding rate of 57%. No significant difference between single and double AI was seen in the study. This study indicates that single or double vaginal insemination with an equal total number of frozen-thawed spermatozoa (400 × 106) can give acceptable fertility results in Norwegian Dairy goats. However, studies on reducing sperm numbers are called for to allow AI donor bucks to be used to their fullest potential.

High rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation - Corrected Proof

2010-06-07

Abstract: In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP (pCX-enhanced green fluorescent protein gene) plasmid and injected into metaphase II oocytes, which were then treated with ionomycin (Io), before further activation with the following agents: 6-dimethylaminopurine (Io-DMAP), additional Io plus DMAP (2Io-DMAP), Io alone (2Io), ethanol (Io-EtOH), or strontium chloride (Io-SrCl2). Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 (Day 0 = ICSI), blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All (100%) of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60% of EGFP-negative embryos (>4 cells) had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments (25.9, 18.7, 14.7, 9.4, and 10.9% respectively; P < 0.05). Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH (52.3, 53.0, 42.8, 28.2, and 29.4% respectively; P < 0.05). Over 80% of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development.

No shortcuts to pig embryonic stem cells - Corrected Proof

T.A.L. Brevini, G. Pennarossa, F. Gandolfi — 2010-06-07

Abstract: The establishment of embryonic stem cell (ESC) lines in domestic species could have great impact in the agricultural as well as in the biomedical field. In particular, derivation of pig ESC would find important applications aimed at improving health and production traits of this species through genetic engineering. Similarly, the immunological, morphological, physiological, and functional similarities to the human make the pig a very effective and suitable animal model for biomedical studies and pre-clinical trials. While proven blastocyst-derived mouse and human ESC lines have been established, no validated porcine ESC (pESC) lines are available. In the present manuscript we briefly discuss some of the factors that make the establishment of ESC lines in the pig, and in animal species other than mouse and human, a very slow process. The paucity of information related to morphology, pluripotency markers, differentiation capability hampers a thorough evaluation of the validity of putative lines.These difficulties are further increased by the lack of reliable antibodies, reagents, and in vitro culture systems that could ensure reliable results in the pig and allow for the screening and long-term maintenance of pESC.Data from the literature suggest that similar regulatory pathways are likely to exist among different species. Coupling of these pathways with their distinct expression patterns, the relative concentrations of pluripotency-related molecules, and timing of embryo development, along with supportive micro-environmental conditions, would appear to vary in a species-specific manner. We feel that the understanding of these subtle but meaningful diversities may provide beneficial information about the isolation of genuine porcine embryonic stem cells.

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell® extender - Corrected Proof

S. Akhter, M.S. Ansari, B.A. Rakha, S.M.H. Andrabi, S. Iqbal, N. Ullah — 2010-06-07

Abstract: This study was designed to compare commercially available extender Bioxcell® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 106 spermatozoa/ml) in tris-citric egg yolk or Bioxcell® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.