Two experiments were conducted to investigate the role of relatively lesser and greater progesterone (P4) concentrations during early follicular development on ovulatory follicle growth and pregnancy rate in beef cattle. In Experiment 1, time of ovulation was synchronized with the 5 d CO-Synch + CIDR (Controlled Internal Drug Release) program in multiparous cows (n = 241). Six days after the 2nd GnRH injection of the pre-synchronization program (d 0), ablation of follicles ≥ 5 mm in the ovaries was performed and cows were assigned to receive either a previously used CIDR and 2x-25 mg PGF2α doses 8 h apart (LoP4), or a new CIDR (HiP4). On d 5, CIDR were removed from all cows, 2x-25 mg PGF2α were administered, and estrous detection tail paint was applied. Timed artificial insemination (TAI) was performed on d 8. On d 5, P4 concentrations were greater (P < 0.01) in the HiP4 (4.9 ± 0.13 ng/mL) than LoP4 (1.0 ± 0.06 ng/mL) treatment group. Conversely, d 5 estradiol (E2) concentrations and follicular diameter were greater (P < 0.01) in the LoP4 (5.0 ± 0.23 pg/mL and 8.9 ± 0.20 mm) than HiP4 (1.5 ± 0.12 pg/mL and 7.4 ± 0.15 mm) treatment group. Follicular diameter at TAI (12.0 ± 0.12 mm, Table 1) and TAI pregnancy rate did not differ (P > 0.10) between treatment groups. In Experiment 2, a new follicular wave was induced with estradiol benzoate on d -7, and cows (n = 275) were assigned on d 0 to receive 25 mg PGF2α and either have the CIDR replaced with a new CIDR (HiP4) or the used CIDR was left in place (LoP4).Furthermore, all cows received GnRH on d 0. The CIDRs were removed from all cows on d 5 and two doses of -25 mg PGF2α were administered. Estrous detection combined with AI 12 h later (Estrus-AI) was performed for 60 h after CIDR removal with TAI coupled with GnRH administration at 72 h if estrus was not detected. The concentrations of P4 on d 5 were greater (P < 0.01) in the HiP4 (2.8 ± 0.10 ng/ml) than LoP4 (1.7 ± 0.05 ng/mL) treatment group. For cows that were detected in estrus after PGF2α administration, estrous response (83.5%) and interval to estrus (55.0 ± 0.5 h) did not differ between treatment groups. Pregnancy rate (combined Estrus-AI and TAI) that resulted from breeding at the time of the synchronized time of estrus was similar between treatment groups (HiP4: 77.1%; LoP4: 82.3%). In conclusion, differences in P4 concentrations during early follicular development do not effect pregnancy rate in beef cows when the cows are inseminated at the time of a synchronized estrus if the cows have similar intervals of proestrus.

Alpacas can only produce one offspring per year. In order to accelerate the genetic gain of a herd, superovulation and embryo transfer can be used to produce multiple embryos from superior females. We hypothesized that the use of dual siring with superovulation would result in the production of multiple embryos sired by different males. After administration of the superovulation protocol, receptive females were bred to two proven males (A and B) 8-12 h apart and ovulation was induced by gonadotropin at the time of the first breeding. Growth of multiple dominant follicles was successfully achieved in 95% of cycles. Females that were receptive after FSH treatment and were bred with both males (order A-B or B-A). Embryo collections were performed 8-9 days post breeding on 15 cycles and 73% of collections recovered >= 1 embryo. A total of 46 embryos, were recovered for an average of 3.13 +/- 3.1 (range 0-10) embryos/flush. Parentage analysis was performed for 23 embryos (6 from A to B, 17 from B to A). Twenty-two of the 23 embryos were determined to be sired by male B, being six embryos from breeding A-B and 16 embryos from breeding B-A. A single embryo from breeding B-A was sired by male A. In conclusion, FSH administered at decreasing doses can be used to promote superovulation resulting in collection of multiple embryos per cycle. However, slight differences in male fertility may affect the frequency of embryos sired by each male.

Objective was to investigate the effect of different progesterone (P4) concentrations during early follicular development on luteinizing hormone (LH) secretion and oocyte characteristics in beef cows. Primiparous cows (n = 24) were estrous pre-synchronized and follicular ablation was performed (d 0) 6 days following the time of ovulation. At the time of follicular ablation, cows were assigned to either: 1) high P4 treatment - HiP4; a new CIDR was inserted on d 0 to supplement P4 from the existing corpus luteum [CL], or 2) low P4 treatment - LoP4; a previously-used CIDR and two doses of PGF 8 to 12 h apart were given on d 0. Concentrations of P4 were greater (P < 0.01) in the cows of the HiP4 than LoP4 group on d 1.5, 2.5, and 3.5. Peripheral concentrations of E2 were greater (P < 0.05) in the cows of the LoP4 than HiP4 group on d 2.5 and 3.5. Frequency of LH pulses was greater (P < 0.05) in the LoP4 than HiP4 group on d 2.5, but mean LH concentration and pulse amplitude did not differ between treatments. Number of follicles aspirated per cow, total oocytes recovered, recovery rate, percentage of oocytes graded 1 to 3, oocyte diameter, percentage BCB+ oocytes, and relative abundance of oocyte mRNA for FST did not differ (P > 0.10) between treatments. In conclusion, lower P4 concentrations during early follicular development resulted in increased LH pulse frequency and E2 concentrations, but did not affect characteristics of oocyte developmental competence.

The present study utilized a 2×2×2 factorial design examining age (old vs. young), follicle size (≥2mm vs. <2mm) and media supplementation (with or without fetal bovine serum [FBS]) to determine factors that might affect in vitro maturation of alpaca oocytes. We hypothesized that oocytes collected from follicles ≥2mm from young alpacas and incubated in maturation media supplemented with FBS would have greater maturation rates than those incubated in any other factorial combination. Oocytes were collected from the ovaries of 11 young alpacas (<10 years old) and 14 old alpacas (>11 years old). Oocytes were classified as morphologically normal oocytes (MNO) and deemed suitable for incubation if ≥3 compact layers of cumulus cells and a homogeneous, evenly granulated cytoplasm were observed. Oocytes from each group of follicle sizes were incubated separately and halves of each group were randomly divided and incubated 24h in chemically defined maturation media with or without 10% FBS. Maturation was defined as the visualization of a polar body at the end of the incubation period. Overall, a greater proportion of MNO were collected from follicles ≥2mm than that obtained from smaller follicles, 55% (136/247) vs. 29.6% (162/547), respectively (P<0.05). A greater proportion of oocytes reached maturation when collected from ≥2mm follicles 36% (49/136) than from <2mm follicles 8% (13/162) (P<0.05). For oocytes obtained from ≥2mm follicles of old alpacas, a greater proportion reached maturation when incubated in media supplemented with FBS than when incubated without FBS; 57.6% (19/33) vs. 18.2% (6/33), respectively (P<0.05).

REASONS FOR PERFORMING STUDY:Placentitis is a prevalent cause of abortion, premature delivery and neonatal death in mares. Early diagnosis is paramount for the survival of the fetus and delivery of a live foal.OBJECTIVES:To determine: 1) Serum amyloid A (SAA) profile in healthy mares during late gestation; 2) if placentitis affects SAA concentrations and 3) the effects of therapy on SAA concentrations and pregnancy outcome in mares with placentitis.METHODS:In Experiment I, 15 healthy pregnant mares were evaluated from 280 days of gestation to 60 h post partum. In Experiment II, pregnant mares were inoculated intra-cervically with Streptococcus zooepidemicus (Day 280-295) and assigned to control (n = 5) and treatment (n = 9) groups. Treatment was initiated at the onset of clinical signs. Serum amyloid A concentrations were determined prior to inoculation and then weekly until abortion or delivery.RESULTS:Serum amyloid A remained at low concentrations (95% confidence interval [CI]: 3.2-8.1 mg/l) during late gestation followed by a significant increase within 36 h post partum; SAA returned to basal concentrations by 60 h post partum. In Experiment II, SAA significantly increased within 96 ± 56 h of inoculation in control mares followed by abortion. Therapy was effective (P<0.05) in preventing the rise in SAA in 66% (6/9) of mares and only one out of 3 mares with increased SAA aborted. Overall, the incidence of abortion was higher in mares with increased SAA concentrations (75%; 6/8) compared with mares in which SAA remained at baseline concentrations (0/6).CONCLUSIONS:Mares with placentitis had significant increased SAA within 96 h post inoculation and concentrations remained increased until abortion in untreated mares. Successful treatment either prevented the rise of SAA concentration or decreased its concentration to baseline concentrations, followed by delivery of a live foal.POTENTIAL RELEVANCE:Serum amyloid A may be used as a prognostic indicator in cases of ascending placentitis in the mare.

Recent research has shown that melatonin has positive effects on sperm viability during cryopreservation. Therefore, melatonin could potentially be included in commercial stallion semen extender to aid in the survival and function of fresh cooled spermatozoa. The objective of this study was to determine effects of melatonin on stallion sperm motility and viability during 48 h of storage at 5°C. In 4 separate trials, ejaculates from 3 stallions were collected and diluted to a final concentration of 500 million sperm/ejaculate with a skim milk-based extender, without antibiotics, supplemented with 0, 0.1, 1.0 or 10.0 mM of melatonin and stored at 5°C for 48 h. Total motility (TM), progressive motility (PM), track velocity (VCL), straight line velocity (VSL) and smoothed path velocity (VAP) were evaluated by Computer-Assisted Semen Analysis (CASA) at 0, 24 and 48 h of storage. An eosin-nigrosin stain was used to subjectively evaluate the live/dead ratio of spermatozoa at 0, 24 and 48 h of storage. Data were analyzed using the PROC MIXED procedure of SAS. TM, PM and sperm viability decreased over time (P < 0.05); however, there were no differences in TM and PM due to melatonin concentrations at any time point. At 48 h, the addition of 10 mM of melatonin to the extended semen had significantly higher PM (P < 0.05). At 24 and 48 h, semen extended with 10mM of melatonin had significantly decreased VCL and VAP compared with control (P < 0.05). VSL and VAP decreased from 0 to 24 h, but there were no differences in VSL between 24 and 48 h. Overall, addition of melatonin to stallion semen extender did not affect sperm motility and viability during storage at 5°C in this study.

The objectives were to compare: (1) preovulatory serum LH concentrations, and (2) synchronization of ovulation, after im or iu administration of the second GnRH treatment of Ovsynch in lactating dairy cows. Lactating cows (N = 23) were presynchronized with two injections of PGF2α given 14 days apart (starting at 34 ± 3 days in milk), followed by Ovsynch (GnRH-7 d-PGF2α-56 h-GnRH) 12 days later. At the time of the second GnRH of Ovsynch (Hour 0), cows were blocked by parity and randomly assigned to 1 of 3 groups: (1) control group (CON; N = 7) were given 2 mL sterile water im; (2) intramuscular group (IM; N = 8) received 100 μg of GnRH im; and (3) intrauterine group (IU; N = 8) had 100 μg GnRH infused in the uterus (2 mL). Blood samples for serum LH concentrations were collected at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Furthermore, ultrasonography was performed twice daily (12-h intervals) from Hours 0 to 60 to confirm ovulation. The LH concentrations were greater (P < 0.05) in the IM than IU and CON groups at Hours 0, 0.5, 1, 1.5, 2, 3, and 4. Although LH concentrations were numerically higher in the IU group, LH concentrations within the IU and CON groups did not change over time. More cows ovulated in the IM (8/8) and IU (7/8) groups within 60 h after the second GnRH administration compared with the CON (2/7) group. In summary, serum LH concentrations were lower in the IU versus IM group, but the proportion of cows that ovulated within 60 h was similar between these two groups. Therefore, iu administration of GnRH may be an alternative route of delivery to synchronize ovulation in beef and dairy cattle.

Serum amyloid A (SAA) and Haptoglobin (Hp) are acute phase proteins, produced during inflammation, such as placentitis. In horses, SAA and SAA1 are protein coding genes. Objectives were to analyze SAA and Hp concentrations and relative abundance of SAA, SAA1 and Hp mRNA transcript in maternal and fetal tissues after experimental induction of placentitis or mares of a control group. Serum Amyloid A family proteins were in marked abundance in the stroma of the endometrium and chorioallantois associated with inflammatory cells. Maternal plasma SAA concentrations were greater (P = 0.01) in mares with experimentally induced placentitis compared to those of the control group. Maternal Hp from the groups were not different, but fetal Hp concentrations of mares with experimentally induced placentitis were greater (P = 0.02). Maternal plasma SAA and Hp concentrations were greater than fetal plasma concentrations in mares with experimentally induced placentitis (P < 0.05). Relative abundance of SAA mRNA transcript was greater in the maternal, fetal liver and chorioallantois of mares with experimentally induced placentitis (P < 0.05) compared to those in the control group. Interestingly, relative abundance of SAA1 mRNA transcript was greater in the chorioallantois of mares with experimentally induced placentitis (P < 0.05). The SAA and Hp concentrations, therefore, were greater in mares with induced placentitis. Furthermore, relative abundance of SAA1 mRNA transcript is specifically greater in the chorioallantois of mares with placentitis, which warrants further studies to elucidate the immunological response of SAA1 in the chorioallantois of mares with placentitis.

REASONS FOR PERFORMING STUDY:Knowledge of commonly encountered fungi infecting the mare's reproductive tract and their respective drug susceptibilities should improve treatment efficacy in mares with fungal endometritis. This is particularly important when practitioners need to start empiric treatment before culture results are complete. OBJECTIVE:To report the spectrum of fungal isolates from uterine samples from mares with reproductive problems and their respective antifungal susceptibilities. METHODS:Equine uterine samples submitted to the Cornell University Animal Health Diagnostic Centre for fungal culture between July 1999 and June 2011 were reviewed. Each mare's reproductive history, fungal culture results, antifungal susceptibilities and concurrent aerobic culture results were evaluated. Patterns of antifungal susceptibility and resistance were assessed over time. RESULTS:One hundred and two fungal isolates were cultured from 92 uterine samples from mares with reproductive problems. Yeast (69%) and mould with septated hyphae (26%) were the most common isolates. Ninety-five to 100% of all fungal isolates were susceptible to the polyenes, while response to the azoles varied with 47-81% of fungal isolates displaying susceptibility. Yeast isolates were 100% susceptible to the polyenes and least susceptible to miconazole (48%) while isolates of mould with septated hyphae were most susceptible to natamycin (100%) and least susceptible to fluconazole (0%). From July 1999 to June 2005 and July 2005 to June 2011, yeast demonstrated increasing resistance to miconazole, while mould with septated hyphae demonstrated increasing resistance to ketoconazole. CONCLUSIONS AND CLINICAL RELEVANCE:Results from this study suggest that polyenes are effective against uterine fungal isolates in vitro and may be the empiric treatment of choice for fungal endometritis. Isolate resistance to specific azoles increased over time.

The objective of this study was to determine the effects of meclofenamic acid, a nonsteroidal anti-inflammatory, on luteal function of beef cattle. A total of 18 Angus cows, aged between 2 and 3 years old, were enrolled in the experiment. All cows were synchronized using a 5-day CIDR protocol. Briefly, cows received 100 μg of gonadorelin diacetate tetrahydrate (GnRH; Cystorelin®, Merial, Athens, GA, USA) and a controlled internal drug release insert (CIDR; Eazi-BreedTM CIDR®, Pfizer Animal Health, New York, NY, USA). Five days later, the CIDR was removed and 50 mg of dinoprost (Lutalyse®, Pfizer Animal Health) was administered intramuscularly. Oestrus was determined by twice daily observations of mounting behaviour and tail painting scores (day of oestrus = Day 0). At 72 h after dinoprost, a second dose of gonadorelin (100 μg, IM) was administered. On Day 14, cows were randomly assigned to the following treatment groups: 1) control: 10 mL of saline solution administered IM; 2) systemic: 2 g of meclofenamic acid administered IM; and 3) oral: 2 g of meclofenamic acid administered orally. Cows were treated once daily for 11 days (i.e. until Day 24) and no adverse reactions were observed. Blood sampling and ovarian ultrasonography were performed every 72 h from Day 0 until Day 12 and then every 48 h until the end of the study. Serum progesterone concentrations were determined by radioimmunoassay and were used to determine functional luteolysis (i.e. progesterone <1 ng mL–1). Ovaries were evaluated for the presence of a corpus luteum and to evaluate follicular growth and subsequent ovulation. One-way ANOVA was used to compare the day of peak progesterone concentration, lifespan of the corpus luteum and the length of the oestrous cycle between groups. Significance was set at P < 0.05 and data are presented as means ± standard error of the mean (Table 1). There were no effects of meclofenamic acid administration on any of the parameters evaluated (P > 0.05). In conclusion, meclofenamic acid administration did not affect luteal function in our study. Potentially, higher doses of meclofenamic acid may be necessary to inhibit prostaglandin synthesis and prevent luteolysis. Table 1.Effects of meclofenamic acid administration on the day of peak progesterone concentration, lifespan of the corpus luteum (CL) and the length of oestrous cycle in beef cows (mean ± standard error of the mean) Funding was provided by The Ohio State University, College of Veterinary Medicine, USDA-Animal Health Formula Funds. The authors are also grateful to the staff at the OSU Beef Center Facility for helping with animal handling and care.

Magnetic-activated cell sorting (MACS) has been used successfully in humans to remove apoptotic sperm from the ejaculate. Annexin V-conjugated microbeads recognise sperm with externalized phosphatidylserine, which is considered one of the features of apoptosis, and the labelled sperm is separated by MACS. The goals of the study were to determine if MACS can be used to separate apoptotic sperm from the ejaculate of stallions; and to determine if removal of apoptotic sperm improves the quality of stallion sperm. Our hypothesis was that MACS would improve semen quality by removing apoptotic sperm, resulting in samples with higher motility and viability. Two ejaculates from three different stallions of good fertility were used. Sperm were diluted with Tyrode’s albumin lactate pyruvate (TALP) and incubated with annexin V-conjugated microbeads for 15 min at 37°C. Control samples were incubated in the absence of annexin V microbeads. The suspension was then loaded into the separation column containing iron globes, which were fitted in a magnet (MiniMACS; Miltenyi Biotec Inc., Auburn, CA, USA). The effluent sample containing annexin-negative sperm was collected and then, the column was removed from the magnetic field and rinsed with TALP to collect the annexin-positive cells. Sperm viability, motility, morphology and caspase activation were determined in all three samples: control, annexin-negative, and annexin-positive. Data were evaluated by ANOVA and individual comparisons were performed by Tukey’s hsd test. Significance was set at P < 0.05 and data is presented as means ± SEM (Table 1). The main effect of stallion was significant only for sperm motility parameters. Sperm recovery rate following MACS was 46 ± 3%. In conclusion, the use of MACS was effective in removing apoptotic sperm from the ejaculate. The annexin-positive population displayed a higher proportion of sperm with activated caspases and lower membrane integrity and motility. However, removal of apoptotic sperm from the ejaculate did not improve sperm parameters in the annexin-negative group compared to control group. In addition, sperm morphology was not affected by MACS. Further studies are necessary to determine if MACS could be used successfully to improve sperm quality from subfertile stallions and frozen semen. Table 1.Sperm parameters following annexin V MACS (mean ± SEM) The authors are thankful to Mark Williams at Miltenyi Biotec Inc. for providing supplies; and Dr Ashok Agarwal at The Center for Reproductive Medicine, Cleveland Clinic, for scientific input.

REASONS FOR PERFORMING STUDY As mule production is often concentrated in remote areas of the world, a simplified semen cryopreservation protocol is required. AIM To compare the seminal parameters of cryopreserved donkey semen in lactose-EDTA and lactose-yolk extenders and the fertility rates on horse mares. METHODS TRIAL 1: Sperm total and progressive motility, vigour (scale 0-5), morphology (major and minor defects) and plasma membrane integrity (HOST) were evaluated in 25 ejaculates from 5 donkey jacks immediately after collection (raw), after chilling to 5°C (chilled) and after freezing/thawing. The semen was mixed with skimmed-milk extender, centrifuged, and then re-suspended in lactose-EDTA or lactose-yolk extender. Semen was loaded into 0.5 ml straws and chilled to 5°C for 1 h, after which samples were either evaluated (chilled semen) or placed above liquid nitrogen for 20 min prior to immersion. Seminal parameters were evaluated by ANOVA and Tukey's test. TRIAL 2: Cryopreserved semen from 3 males was used to inseminate 53 mares at 60 oestrous cycles randomly assigned to lactose-yolk (n = 30 cycles) or lactose-EDTA (n = 30 cycles) extenders. Pregnancy diagnosis was performed 15 and 25 days post ovulation. The pregnancy rates were compared using Chi-squared tests. RESULTS TRIAL 1: No significant differences were evident in any seminal parameters between extenders after either chilling or cryopreservation. Total and progressive motility were significantly (P<0.05) lower in cryopreserved semen than raw and chilled semen for both extenders. TRIAL 2: Pregnancy rates did not significantly differ between extenders (lactose-EDTA extender 53.33 and 43.33%; lactose-yolk 50.0 and 46.66% for Days 15 and 25 post ovulation, respectively). CONCLUSIONS Cryopreservation of donkey semen using the simplified lactose-yolk extender resulted in similar seminal parameters and fertility rates when compared to lactose-EDTA extender. POTENTIAL RELEVANCE Lactose-yolk extender may be advocated as a simple, easy to prepare extender, for use in geographically isolated enterprises producing mules throughout the world.

The use of assisted reproductive techniques (ART) has helped owners to produce offspring from valuable mares that were considered infertile using standard breeding techniques. Before referring a mare for an ART, the practitioner should be able to identify the underlying cause of subfertility of the mare. The objective of this review is to provide information regarding embryo transfer, oocyte transfer and intracytoplasmic sperm injection, the three most common ART used in equine practice. Knowing the complexity as well as the risks of these techniques, enables practitioners to refer a subfertile mare to the least complex and most appropriate and successful ART that can overcome specific causes of infertility.

Glutathione (GSH), an important bioactive product, is widely used in production of pharmaceuticals and foods. In this study, four different vector systems, pET28a, pUC18, pUC19-P32, and pUC19-Pabb, were applied for expression of gshF, encoding the bifunctional glutathione synthetase of Streptococcus thermophiles. These four constructs were named as pET28a-gshF, pUC18-gshF, pUC19-P32-gshF and pUC19-Pabb-gshF, respectively, and then introduced into Escherichia coli strain BL21(DE3) for further investigation of protein expression and GSH production. The expression levels of the GshF in BL21(pUC19-P32-gshF) and BL21(pUC19-Pabb-gshF) were much lower than those of BL21(pET28a-gshF) and BL21(pUC18-gshF). In the fed-batch fermentation, the GSH accumulated by BL21(pUC18-gshF) reached 15.21 g/L, which was the highest level of GSH biosynthesis ever reported. Although BL21(pUC19-Pabb-gshF) produced less GSH compared to BL21(pUC18-gshF), the final GSH concentration produced by BL21 (pUC19-Pabb-gshF) still accumulated to 5.09 g/L, which indicated the potential application of the constitutive promoter in GSH production.

Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2±0.4 yr of age (mean±S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0±0.1h after administration of hCG to estrous donors and cultured 16.4±0.2h prior to transfer into recipients’ oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P>0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than ≥20 yr (mean ages±S.E.M. of 15.5±0.4 and 23.0±0.3 yr, respectively) tended (P=0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.

The objectives were to compare embryo development rates after oocyte transfer with: (1) intrauterine or intraoviductal inseminations of fresh semen versus intraoviductal insemination of frozen semen; (2) intraoviductal versus intrauterine inseminations of cooled semen. In Experiment I, oocytes were transferred into the oviduct, and recipients were inseminated into the uterus with 1×109 fresh spermatozoa, or into the oviduct with 2×105 fresh or frozen-thawed spermatozoa. In Experiment II, semen was cooled to 5 °C before intrauterine insemination with 2×109 spermatozoa or intraoviductal inseminations of 2×105 spermatozoa (deposited with the oocytes). In Experiment I, embryo development rates were similar (P>0.05) for intrauterine versus intraoviductal inseminations when fresh semen was used (8/14, 57% and 9/11, 82%, respectively). However, embryo development rates were lower (P<0.05) when frozen spermatozoa were placed within the oviduct (1/12, 8%). In Experiment II, embryo development rates were higher (P<0.05) when cooled semen was used for intrauterine (19/23, 83%) versus intraoviductal (4/16, 25%) inseminations. We concluded that intraoviductal insemination can be successfully performed using fresh spermatozoa. However, the use of cooled and frozen spermatozoa for intraoviductal inseminations was less successful, and needs further investigation.

Transportation of equine ovaries would allow shipment of oocytes for research purposes or transfer after the death of a valuable mare. The objective of this study was to compare two temperatures for maintaining ovaries during a transport interval of 18-24 h. The goal was to obtain pregnancies after transport of ovaries, maturation of oocytes in vitro, and transfer of oocytes. Each shipment was composed of ovaries four to seven mares collected from an abattoir. From each mare, one ovary was packaged at approximately 12 degrees C, and the other was packaged at approximately 22 degrees C. Upon arrival at our laboratory, oocytes were collected and cultured for 24 h. For each transfer, between 9 and 15 oocytes from each group were placed into the oviducts of estrous mares through standing flank laparotomies. Recipients received human chorionic gonadotropin (hCG; 2000 IU, i.v.) 30-36 h before transfer (to synchronize ovulation). Recipients were inseminated 18-20 h before transfers with 2 x 10(9) progressively motile sperm. Uteri of recipients were examined with ultrasound to determine the number of developing embryos. On Day 16 ( ovulation = day 0), developing embryos were recovered by uterine lavage. Parentage verification was performed on recovered vesicles. Pregnancy rates were analyzed by Chi-square. The percentage of oocytes that developed into embryonic vesicles on Day 16 was not different between transport temperatures (22 degrees C, 13/73, 18% versus 12 degrees C, 11/73, 15%). In conclusion, pregnancies were obtained from in vitro matured oocytes that were recovered from ovaries transported for 18-24h at 12 or 22 degrees C.

The ability of sperm to bind to zonae pellucidae (ZP) has been correlated with fertilizing capacity of sperm in several species. Limited numbers of equine oocytes are available to perform such assays. Therefore, use of heterologous ZP to perform gamete binding tests with stallion sperm would be useful. We have found that addition of 10% of skim milk-based extender with glucose [EZ-Mixin®, Animal Reproduction Systems, Chino, USA;; (EZ)] to TALP significantly increased the number of stallion sperm bound to bovine ZP. Objectives of the present experiments were to determine: (1) if stallion sperm bind in similar numbers to equine and bovine ZP, and (2) the effects of skim milk, milk proteins and glucose on sperm binding to ZP. Denuded bovine (immature) and equine (mature) oocytes were stored at 5°C in salt solution (1.5M MgCl2, 40mM HEPES, 0.1% PVP). In Experiment I, 4 ejaculates from 2 stallions were centrifuged at 300g for 6min, and sperm pellets were resuspended in 1mL of TALP or EZ. Sperm were stained with Hoechst 33342, centrifuged, and resuspended to 2×106 sperm mL−1. Oocytes were placed into droplets of 45μL of TALP (7 to 10 oocytes/trt/ejac). Extended sperm (5μL) were added to oocytes, resulting in 2times105 sperm mL−1, and the mixutre was incubated for 2h at 38.5°C. Oocytes then were pipetted in TALP to remove loosely attached sperm and observed with fluorescence microscopy;; mean numbers of sperm bound to bovine and equine ZP for TALP were 29±1.9 and 36±2.6 (P>0.1) and for EZ, 149±5 and 152±6.3 (P>0.1), respectively. More sperm bound to ZP with EZ than to ZP with TALP (P<0.001). Experiment II used 4 ejaculates from 4 stallions. After initial centrifugation, sperm were resuspended in 1mL of each of six extenders: TALP, EZ, TALP containing 89.5mM glucose (TG), TALP containing 163.5mM glucose (THG), TALP containing 2.4mgmL−1 of skim-milk powder (TSM), and INRA 96® (IMV Technologies, L’Aigle, France) that contains 27mgmL−1 of native phosphocaseinate. Hoechst 33342-stained sperm and bovine oocytes were processed as described for Experiment I. Treatments containing milk proteins resulted in more sperm binding (P<0.01) than those without milk proteins (Table 1). In conclusion, use of bovine oocytes led to similar results for equine and bovine oocytes;; therefore, bovine oocytes can be used for binding assays with stallion sperm. High concentrations of glucose increased numbers of sperm bound to ZP;; however, presence of milk or milk proteins was more effective in enhancing binding of sperm to ZP. INRA96 contains relatively low glucose (67mM) and one milk protein. Therefore, we hypothesize that native phosphocaseinate may cause increased sperm binding to ZP. Table 1 Mean sperm bound per ZP±SEM (n=38–40/group)

Progesterone (P4), 17β- estradiol (E2) and androstenedione (A4) plasma concentrations were correlated with palpated corpora lutea (CL), recovered embryos and viable embryos in 13 Nelore cows induced to superovulate with FSH, starting on Day 10 of the estrous cycle. Administration of FSH increased the number of ovulations and recovered embryos. Plasma P4, E2 and A4 levels on Day 0 and of P4 on Days 10 and 11 of the cycle were not correlated with the superovulatory response. Determination of CL by palpation per rectum was used to estimate the number of recovered embryos. Plasma P4 levels higher than 1 ng/ml on the induced estrus day (Day 14) had an adverse effect on the embryo viability rate. Plasma E2 concentrations on Day 14 were positively correlated with the number of viable embryos collected, a correlation that has not been previously reported. The present data indicate that plasma P4 and E2 concentrations in FSH-PGF2α-treated Nelore cows are useful for the identification of 2 different populations of Nelore donors and are correlated with superovulatory response and, particularly, with the number of viable embryos.