Factors impacting equine sperm recovery rate and quality following cushioned centrifugation.

Two experiments were conducted to investigate modifications in cushioned centrifugation of stallion semen. Specifically, the effects of tube type, centrifugation medium, cushion type, and centrifugation force on post-centrifugation sperm recovery rate and quality were evaluated. In Experiment 1, sperm recovery rate was higher (P<0.05) in conventional plastic conical-bottom tubes (103%) than in newly developed glass nipple-bottom tubes (96%) following cushioned centrifugation; however, several measures of semen quality (i.e., % total motility [MOT], % progressive motility [PMOT], curvilinear velocity, and average-path velocity) yielded higher values following centrifugation in nipple-bottom tubes (P<0.05). Sperm recovery rate following cushioned centrifugation was similar between semen previously diluted in optically clear centrifugation extender (100%) and semen diluted in opaque centrifugation extender (100%); however, MOT and PMOT were higher in semen subjected to cushioned centrifugation in opaque extender (P<0.05). An extender by tube-type interaction was not detected for recovery rate or post-centrifugation semen quality. In Experiment 2, sperm recovery rate following cushioned centrifugation in nipple-bottom tubes was similar when forces of 400xg or 600xg were applied (90 and 90%, respectively; P>0.05), and no resulting differences in semen quality were detected between these treatment groups (P>0.05). The type of iodixanol cushion medium used (i.e., OptiPrep, Eqcellsire Component B, or Cushion Fluid did not impact post-centrifugation semen quality, based on the laboratory values measured (P>0.05). In conclusion, cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes yielded a high sperm harvest, while maintaining sperm function. An optically opaque extender, commonly used in the equine breeding industry, can be used to achieve this goal.

Cholesterol-to-phospholipid ratio in whole sperm and seminal plasma from fertile stallions and stallions with unexplained subfertility.

Semen samples were collected from six fertile stallions and seven stallions with unexplained infertility. Percentages of motile sperm (77.5 +/- 11.3 versus 67.5 +/- 12.2, P = 0.2), and progressively motile sperm (70.8 +/- 13.6 versus 60.7 +/- 14.0, P = 0.2) were similar between fertile and subfertile stallions, respectively. Morphologic characteristics in ejaculates of control and affected stallions (% normal: 60.2 +/- 18.2 versus 52.9 +/- 11.3, P = 0.4; % abnormal heads 7.3 +/- 4.8 versus 12.1 +/- 5.0, P = 0.11; and % abnormal acrosomes 1.6 +/- 2.1 versus 3.0 +/- 3.4, P = 0.4) did not differ. After incubation with the calcium ionophore A23187, acrosome reaction rate of sperm from fertile stallions was 96 +/- 2.8% whereas only 2.9 +/- 2.5% of sperm from stallions with unexplained subfertility had acrosome reacted (P < 0.001). Molar amounts of cholesterol and phospholipid in whole sperm and seminal plasma did not differ (P > 0.1) between fertile and subfertile stallions. However, the molar ratio of cholesterol-to-phospholipid was 2.5 times greater in the seminal plasma (P = 0.09) and 1.9 times greater (P = 0.009) in whole sperm of subfertile stallions compared to fertile stallions.

The effect of dual-hemisphere breeding on stallion fertility.

Breeding records were analyzed from 24 Thoroughbred stallions that were subjected to dual-hemisphere breeding (DH), including novice (first-year; NOV; n = 11) and experienced (EXP; n = 13) stallions. Fertility variables included seasonal pregnancy rate, pregnancy rate per cycle, and first-cycle pregnancy rate. In addition, values for book size, total number of covers, distribution of mare type (maiden, foaling, and barren) within a stallion's book, cycles per mare, and mare age were examined. Some data were also categorized by mare type (maiden-M, foaling-F, and barren-B). Five separate analyses of the data were performed. For Analyses 1-3, the effects of hemisphere (northern hemisphere [NH] vs. southern hemisphere [SH]) and breeding order (refers to the first [O1] or second [O2] season within the first year of dual-hemisphere breeding) were examined for all stallions (combined group [CG]), NOV stallions only, and EXP stallions only, respectively. Fertility values were generally higher in the SH than the NH (P < 0.05), whereas book size, total number of covers, and cycles per mare were higher in the NH than the SH (P < 0.05). Book size and total covers were negatively correlated to first cycle pregnancy rate (r = -0.57, r = -0.71, respectively; P < 0.05) for NOV stallions. Pregnancy rate per cycle was also negatively correlated with total covers (r = -0.58; P < 0.05) for NOV stallions. Similar trends were noted for Groups CG and EXP, but the relationship was not as marked as for NOV stallions. The fertility of O1 was generally similar to O2 (P > 0.05). For Analysis 4, fertility of DH breeding seasons was compared to single hemisphere (SIN) breeding seasons within the same 16 stallions and was found to be similar between the two groups (P > 0.05). For Analysis 5, the effect of the number of consecutive DH breeding seasons on fertility was examined and was found to remain unchanged (P > 0.05). In summary, no adverse effects of DH breeding on fertility were detected. Fertility was higher when stallions were bred in the SH, as compared to the NH. Potential reasons for higher fertility achieved in the SH were smaller book sizes and better mare reproductive quality.

Characterisation of lymphocyte subsets in the equine oviduct.

REASONS FOR PERFORMING STUDY: The equine oviduct is the site of fertilisation and location of embryonic development during the first 5 or 6 days. It therefore has an important influence on mare fertility. Although histopathological changes have been described previously, there is limited information regarding lymphocyte subtypes present in the mucosa of the normal equine oviduct. OBJECTIVES: To characterise the distribution of CD3+, CD4+, CD8+ and B lymphocytes in the equine oviduct from inseminated mares during oestrus and dioestrus, and from noninseminated mares during the immediate post ovulatory period. METHODS: Oviductal tissues were collected from noninseminated mares at oestrus (> 30 mm follicle, n = 4), at Day 1 post ovulation (n = 3) and at dioestrus (Day 7 post ovulation; n = 4). Oviducts were also collected from inseminated mares at Days 1, 2, and 3 post ovulation (n = 4 for each period). Cross-sections of tissues from the ampullar-isthmic junction from each oviduct were snap frozen and cryostat sections stained by the immunoperoxidase technique with monoclonal antibodies directed against equine lymphocyte surface markers for B cells as well as CD3+, CD4+ and CD8+ cells. RESULTS: In all oviductal sections examined, B cells were rare whereas T cells were relatively abundant. The predominant cell type found was the CD8+ phenotype, with a lesser number of CD4+ cells. Among mares, individual variation was large; therefore, although breeding status and stage of oestrous cycle appeared to alter lymphocyte populations, these differences were not significant. CONCLUSIONS AND POTENTIAL RELEVANCE: A population of CD3+, CD4+ and CD8+ cells exists within the mucosal region of the equine oviduct. The density of these cells is similar to that described in the human oviduct. Their function is not currently known, but they may be involved with modulation of the maternal response to the presence of spermatozoa or the early conceptus within the equine oviduct. As our capacity to differentiate these cell types improves, along with the ability to identify the specific cytokines they produce, their functional significance will become more apparent.

The effect of two levels of hemospermia on stallion fertility.

Hemospermia can occur consistently or intermittently in stallion ejaculates and may cause a reduction in the fertility of the affected ejaculate. It is unknown what amount of blood in an ejaculate leads to subfertility. This study investigated the effect of higher and lower levels of hemospermia (50% and 5%, respectively) on fertility using 24 reproductively normal mares inseminated over three consecutive estrous cycles with fresh extended semen. Mares inseminated with a 5% blood-contaminated ejaculate became pregnant at the same rate (75% per cycle; 18 of 24) as the mares inseminated with blood-free (control) semen (75% per cycle; 18 of 24). The ejaculates containing 50% blood were sterile (0% per cycle, 0 of 24). We concluded that it is the amount of blood, not the mere presence of blood, in an ejaculate that impacts fertility.

Relationship of seminal plasma level and extender type to sperm motility and DNA integrity.

The relationship between seminal plasma level (0, 10, or 20%) and extender type [Kenney type (EZ-Mixin-CST) or Kenney-modified Tyrodes-KMT] to the susceptibility of sperm DNA to denaturation and sperm motility measures were investigated in cooled (5 degrees C) stallion sperm. Three ejaculates from each of three fertile stallions were collected in an artificial vagina and processed as follows: diluted one part uncentrifuged semen with four parts of extender to a final concentration of 20% seminal plasma in either CST or KMT (20% CST; 20% KMT); diluted to a final concentration of 25 million sperm/mL in either CST or KMT (10% CST; 10% KMT); centrifuged to remove virtually all seminal plasma and resuspended in either CST or KMT (0% CST-Cent; 0% KMT-Cent); centrifuged semen to remove virtually all seminal plasma and resuspended with previously filtered seminal plasma from the same stallion in either CST or KMT to a final concentration of 20% seminal plasma (20% CST-Cent; 20% KMT-Cent). Sperm motion characteristics were determined by CASA and DNA integrity (%COMP, percent of cells outside the main population) evaluated by the Sperm Chromatin Structure Assay prior to cooling, and after 24 and 48 h cooled-storage at 5 degrees C. After 48 h of storage at 5 degrees C, extenders with 0% seminal plasma (0% CST-Cent, 0% KMT-Cent) maintained highest quality DNA (P < 0.05), but 0% KMT-Cent maintained higher velocity measures (P < 0.05) than 0% CST-Cent. Total sperm motility was highest (P < 0.05) in 0% CST-Cent, 0% KMT-Cent, 10% CST, 20% CST-Cent, and 20% CST compared to the other treatment groups. Progressive sperm motility was highest (P < 0.05) after 48 h of storage in the treatment with 10% seminal plasma in Kenney extender (10% CST), despite a reduction in DNA integrity. Regardless of extender type, addition of 20% seminal plasma following centrifugation resulted in almost a two-fold increase in %COMP(alpha t), even though one of the treatments (20% CST-Cent) maintained total and progressive motility similar to treatments with no seminal plasma, suggesting that sperm motility and DNA integrity may respond independently to environmental conditions. Overall, better quality sperm features (motility and DNA) were maintained in sperm from which seminal plasma was removed followed by resuspension in either Kenney extender or modified Kenney Tyrodes-type extender.

Dexamethasone acutely regulates endocrine parameters in stallions and subsequently affects gene expression in testicular germ cells.

Testicular steroidogenesis and spermatogenesis are negatively impacted by stress-related hormones such as glucocorticoids. The effects of two injections of a therapeutic dose of dexamethasone (a synthetic glucocorticoid, 0.1mg/kg; i.v.) given 24h apart to each of three stallions were investigated and compared to three saline-injected control stallions. Dexamethasone decreased circulating concentrations of cortisol by 50% at 24h after the initial injection. Serum testosterone decreased by a maximum of 94% from 4 to 20h after the initial injection of dexamethasone. Semen parameters of the dexamethasone-treated stallions were unchanged in the subsequent two weeks. Two weeks after treatment, stallions were castrated. Functional genomic analyses of the testes revealed that, of eight gene products analyzed, dexamethasone depressed concentrations of heat shock protein DNAJC4 and sperm-specific calcium channel CATSPER1 mRNAs by more than 60%. Both genes are expressed in germ cells during spermiogenesis and have been related to male fertility in other species, including humans. This is the first report of decreased DNAJC4 and CATSPER1 mRNA concentrations in testes weeks after dexamethasone treatment. Concentrations of these mRNAs in sperm may be useful as novel markers of fertility in stallions.

Gonadal and pituitary responsiveness of stallions is not down-regulated by prolonged pulsatile administration of GnRH.

The objective of this study was to determine if prolonged pulsatile administration of homologous gonadotropin-releasing hormone (GnRH) at therapeutic or 5x therapeutic doses would cause down-regulation of the stallion's hypothalamic-pituitary-testicular axis. Fifteen stallions were randomly assigned to three treatment groups (n=5/group) and received a 0.5 ml subcutaneous dose of saline (group 1), 50 microg GnRH (group 2), or 250 microg GnRH (group 3) every 2 hours for 75 days. Weekly evaluations of follicle stimulating hormone, luteinizing hormone, and testosterone and monthly evaluations of daily sperm output and spermatozoal motility failed to demonstrate any decreased pituitary or gonadal responsiveness within or among treatment groups (P > 0.1) as a result of treatment with GnRH. Results of this study demonstrate that the hypothalamic-pituitary-testicularaxis of the stallion, unlike that of other domestic species, is remarkably refractory to GnRH-induced down-regulation.

Lamprey gonadotropin-releasing hormone-III selectively releases follicle stimulating hormone in the bovine.

Recent studies have shown that lamprey gonadotropin-releasing hormone (l-GnRH) is localized in the mammalian brain, and that l-GnRH-III, can selectively induce FSH secretion in the rat both in vivo and in vitro. Consequently, the purpose of this study was to determine if l-GnRH-III could elicit selective FSH release in cattle and compare this response with that to mammalian luteinizing hormone releasing hormone (m-LHRH). Cattle were chosen as the animal model because previous studies have demonstrated that FSH and LH are secreted by separate gonadotropes in that species. For these studies, crossbred cycling heifers were implanted with jugular cannulae and l-GnRH-III was infused either between Days 9-14 or on Day 20 of the estrous cycle. Blood samples were collected both before and following peptide infusion. Our results demonstrate that during Days 9-14 of the estrous cycle (luteal phase), when progesterone levels averaged between 4 and 5 ng/ml, a dose of 0.25 mg of l-GnRH-III induced the release of FSH (P < 0.05), but not LH. A 0.5 mg dose of l-GnRH-III caused a greater release of FSH (P < 0.01), but still did not induce LH release. Higher doses of the peptide were capable of significantly releasing both gonadotropins. Importantly, during the luteal phase, doses of 0.5 and 2 mg of m-LHRH were ineffective in stimulating FSH, but did elicit marked increases (P < 0.001) in LH. Again, progesterone levels averaged 4-5 pg/ml. In order to assess gonadotropin releasing ability of l-GnRH-III at a different phase of the estrous cycle, some animals were administered the peptide on Day 20, when progesterone levels were below 1.0 pg/ml. At this time, the l-GnRH-III induced the release of LH (P < 0.01), but not FSH. Overall, our results demonstrate that l-GnRH-III can selectively induce FSH in cattle during the luteal phase, whereas m-LHRH was ineffective in that regard. Furthermore, the fact that l-GnRH-III can selectively stimulate FSH when serum progesterone is high, and LH when serum progesterone is low, suggests its actions are under strong control of this steroid. We suggest the FSH releasing capacity of l-GnRH-III in cattle could render this peptide useful for enhancement of reproductive efficiency in this species.

Techniques for evaluating selected reproductive disorders of stallions.

Numerous techniques may be used for evaluation of the different reproductive disorders of the stallion. Approaches may vary from real-time ultrasonography and biopsy for evaluating testicular tumors to use of special assays for evaluating sperm or plasma for presence of antisperm antibodies. This communication addresses techniques used to evaluate five relatively uncommon, but perplexing, disorders of breeding stallions: (1) seminal vesiculitis, (2) hemospermia associated with idiopathic urethral defects, (3) acrosomal dysfunction, (4) abnormal spermatozoal chromatin, and (5) azoospermia.

Advances in cooled semen technologies: seminal plasma and semen extender.

This study evaluated motility and fertility of uncentrifuged and centrifuged equine semen following dilution in a skim milk-glucose extender with or without supplemental Tyrode's medium. In addition, the effect of seminal plasma addition to each extender was evaluated. For Experiment 1, motility of 48h cooled, stored spermatozoa was evaluated following eight dilution treatments: uncentrifuged and diluted 1:4 (v/v) in skim milk-glucose extender (EZ Mixin CSTJ; CST-1:4) or in CST supplemented 65:35 (v/v) with modified Tyrode's medium (KMT-1:4); uncentrifuged and diluted to 25x10(6) spermatozoa/ml in CST (CST-1:9) or in KMT (KMT-1:9); centrifuged and diluted in CST with 0% seminal plasma (CST-0) or 20% seminal plasma (CST-20) or centrifuged and diluted in KMT containing 0% seminal plasma (KMT-0) or in KMT containing 20% seminal plasma (KMT-20). Sperm motility parameters evaluated included percentage of total motile sperm (% TMOT), percentage of progressively motile sperm (% PMOT), curvilinear velocity (VCL) and straight-line velocity (VSL). Mean % PMOT was lower (P<0.05) for spermatozoa extended in CST-1:4 compared to CST-1:9, whereas, all motility parameters were reduced (P<0.05) in KMT-1:4 compared to KMT-1:9. Spermatozoa extended in CST-1:4 had greater % TMOT, % PMOT and VSL (P<0.05) than in KMT-1:4. Spermatozoa extended in CST-1:9 had greater (P<0.05) % PMOT than in KMT-1:9, however, VCL was greater (P<0.05) in KMT-1:9. Mean VCL and VSL were lower (P<0.05) for spermatozoa extended in CST-0 compared with CST-20, whereas, spermatozoa extended in KMT-0 had greater (P<0.05) % TMOT, % PMOT and VSL compared to spermatozoa extended in KMT-20. Mean % TMOT and % PMOT were greater (P<0.05) in CST-20 compared to KMT-20, however, KMT-0 increased (P<0.05) velocity measures (VCL and VSL) compared to CST-0. In Experiment 2, fertility of centrifuged spermatozoa diluted in either CST-20 or KMT-0 was similar (P>0.05). We conclude that modified Tyrode's medium was not detrimental to establishment of pregnancy. Use of modified Tyrode's medium may improve spermatozoal motility and pregnancy rates for cooled transport of semen from stallions in which all seminal plasma must be removed because of suspected toxic effects of seminal plasma on spermatozoal viability, however, Tyrode's medium may be detrimental to sperm motility when seminal plasma is present.

Effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage.

The objective of this study was to determine if centrifugation and partial removal of seminal plasma would improve spermatozoal motility in semen from stallions whose whole ejaculates have poor tolerance to cooling and storage. Stallions were divided into two groups (n = 5/group) based on the ability of their extended semen to maintain spermatozoal motility after cooling and storage. Group 1 stallions ("good coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by < or = 30% of progressive motility prior to storage. Group 2 stallions ("poor coolers") produced semen in which progressive spermatozoal motility after 24 h of cooling and storage was reduced by > or = 40% of progressive motility prior to storage. The sperm-rich portion of each ejaculate was divided into 4 aliquots. Two aliquots underwent standard processing for cooled transported semen and were examined after 24 and 48 h of cooling and storage in an Equitainer. The remaining two aliquots were diluted 1:1 with semen extender, then centrifuged at 400 x g for 12 min at room temperature. After centrifugation, approximately 90% of the seminal plasma was removed, and the sperm pellet was resuspended in extender to a final concentration of 25 to 50 x 10(6) sperm/mL. These aliquots were then packaged as for the non-centrifuged aliquots and examined after 24 and 48 h of storage. The spermatozoal motion characteristics in fresh semen and after 24 and 48 h of cooling and storage was determined via computer-assisted semen analysis. Centrifugation and partial removal of seminal plasma increased the percentage of progressively motile spermatozoa and limited the reduction in progressive spermatozoal motility of "poor cooling" stallions after 48 h of cooling and storage. Results of this study indicate that centrifugation and partial removal of seminal plasma is beneficial for stallions whose ejaculates have poor tolerance to cooling and storage with routine semen dilution and packaging techniques, especially if the semen is stored for > 24 h.

In vitro maturation of equine oocytes obtained from different age groups of sexually mature mares.

Oocytes were harvested from mare ovaries obtained at slaughter and were divided into 3 groups based on the age of the donor. The age groups consisted of young (2 to 7 yr), middle-aged (8 to 14 yr) and aged (>or=15 yr) mares. There were no differences between age groups in the proportions of follicles available for examination or the proportions of normal, abnormal or total oocytes collected. After 24 h of culture, the overall maturation rate to the second metaphase (MII) was 52.7%. Maturation rates for oocytes obtained from young and middle-aged mares were similar, but oocytes from aged mares were only approximately 25% as likely to reach MII and they were 3 times more likely to remain at metaphase I. Twelve oocytes had chromosome spreads suitable for counting; 6 were haploid, 2 were hyperhaploid and 4 were hypohaploid. Insufficient numbers of readable spreads precluded comparisons of chromosome complements between age groups.

Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa.

This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loaded, they were subjected to one of three ambient storage temperatures: 1) 22 degrees C for 72 h, 2) -20 degrees C for 6 h followed by 22 degrees C for 66 h, or 3) 37 degrees C for 72 h. Cooling rates and storage temperatures of semen extender in each container were monitored with thermocouples and a chart recorder. In Experiment 2, semen from each of three stallions (3 ejaculates per stallion) was diluted to 25 x 10(6) spermatozoa/mL with semen extender, divided into 40 mL aliquots and loaded into transport containers as in Experiment I. Containers were subjected to one of three ambient storage conditions: 1) 22 degrees C for 24 h, 2) -20 degrees C for 6 h, followed by 22 degrees C for 18 h, or 3) 37 degrees C for 24 h. After 24 h of storage, spermatozoal motion characteristics (percentage of motile spermatozoa; MOT, percentage of progressively motile spermatozoa; PMOT, and mean curvilinear velocity; VCL) were evaluated using a computerized spermatozoal motion analyzer. Significant interactions were detected among storage conditions and semen transport containers for the majority of the temperature endpoints measured. When exposed to temporary ambient freezing conditions, the lowest temperatures attained by samples in containers ranged from -2.8 to 0.8 degrees C. Lowest temperature samples attained was not correlated (P > 0.05) with spermatozoal motility under any ambient condition. However, time below 4 degrees C was highly correlated (P < 0.05) with a reduction in spermatozoal motility. Mean cooling rates from 20 degrees C to 8 degrees C did not correlate with spermatozoal motility, except when containers were exposed to temporary freezing conditions. No container cooled samples below 6 degrees C in 22 degrees C or 37 degrees C environments except for the ExpectaFoal, in which samples fell below 4 degrees C under all ambient conditions. Ambient temperature affected MOT, PMOT and VCL of semen stored in all containers (P < 0.05) except for the Equitainer II in which motion characteristics remained high and were similar among all ambient temperatures (P > 0.05). Results suggest that stallion semen may be able to tolerate a wider range of cooling rates and storage temperatures than previously considered safe.

Effects of ecbolic agents on measurements of uterine involution in the mare.

Thirteen postparturient mares were used to investigate the effects of ecbolic agents on the rate of uterine involution. Mares were randomly assigned to one of three treatment groups: Group S = intravenous injection of 2 ml saline twice daily for 10 days post partum (n=4); Group O = intravenous injection of 20 units oxytocin twice daily for 10 days post partum (n=4); and Group P = intramuscular injection of 500 mcg fluprostenol twice daily for 10 days post partum (n=5). Ovulation was determined by daily transrectal ultrasonographic examination of the ovaries. On Days 6, 11 and 16 post partum, transrectal ultrasonography was used to measure cross-sectional diameters of the uterine body, uterine horns and fluid within the uterine lumen. Uteri were swabbed for aerobic bacteriologic culture on Days 11 and 16 post partum. Uterine biopsies were obtained from the base of the previously gravid uterine horn on Days 11 and 16 post partum for subjective assessment of endometritis and morphometric analysis of endometrial histoarchitecture. Mean values for all measurements of uterine involution did not differ among groups (P>0.05). For all mares, the diameter of luminal fluid was not correlated to diameter of the uterine body or uterine horns, or to morphometric measurements of endometrial histoarchitecture of the previously gravid uterine horn (P>0.05). Likewise, accumulation of fluid within the uterine lumen was not associated with endometritis or recovery of potential bacterial pathogens (P>0.05). Mean diameter of the previously gravid uterine horn was negatively correlated with morphometric measures of endometrial histoarchitecture of the previously gravid uterine horn (P<0.01).

Effects of a new injectable short-term release deslorelin in foal-heat mares.

Mares treated with subcutaneous deslorelin implants on the first postpartum estrus early in the breeding season had significant reductions in the number of large follicles at early pregnancy examinations and delayed return to estrus (in mares that failed to become pregnant); these adverse effects were attributed to a prolonged release of the drug from the implant. In 2003, an injectable short-term release (<24 h) deslorelin product became available. The objective of this study was to determine if this product would hasten ovulation in early foaling first postpartum estrus mares without reducing the number of large follicles at early pregnancy examination (14-15 days postovulation). Beginning 5-6 days postpartum, first postpartum estrus (foal-heat) mares were teased daily and examined thrice weekly (Tuesday, Thursday and Saturday) by transrectal ultrasonography. Mares in estrus with a follicle > or = 34 mm diameter on Tuesdays or Thursdays were alternately assigned to: Treatment 1, n = 17; 1.5 mg injectable short-term release deslorelin, or Treatment 2, n = 16; Control (no treatment). The schedule allowed accurate determination of the number of mares ovulating within 2 days of treatment (i.e., ovulations detected on Thursday or Saturday). Mares were mated on the day of treatment and at 2-day intervals until either ovulation was confirmed or until behavioral estrus ceased. Transrectal ultrasonography was done 14-15 days after ovulation to assess ovarian follicles and pregnancy status. Fewer covers were required and more mares ovulated within 2 days of treatment in deslorelin-treated versus Control mares (P < 0.01). Pregnancy rates were normal (69%) in deslorelin-treated mares. The number of large follicles 14-15 days after ovulation did not differ between deslorelin-treated and Control mares (P > 0.10), suggesting follicular suppression did not occur with this formulation of deslorelin.

The effect of postbreeding uterine lavage on pregnancy rate in mares.

One stallion and 54 mares were used in an experiment to evaluate the effect of postbreeding uterine lavage on pregnancy rate in mares. All mares were inseminated with 250 x 10(6) progressively motile sperm every other day during estrus until detection of ovulation. Mares (n = 18) were randomly assigned to one of three treatment groups: 1) no postbreeding uterine lavage (control); 2) uterine lavage at 0.5 h postbreeding; or 3) uterine lavage at 2 h postbreeding. A dilute solution of povidone-iodine (PIS; 0.05%) previously determined to render spermatozoa immotile in vitro was used to lavage the mare uteri. One liter PIS, prewarmed to 40 degrees C, was used for each lavage. Pregnancy status of mares was determined at 21 d and 36 d post ovulation, using transrectal ultrasonography. The pregnancy rate of Group 1 (66.7%) was higher than that of Group 2 (22.2%; P<0.05) or Group 3 (33.3%); P<0.10). The pregnancy rates of Groups 2 and 3 were similar (P>0.70). Evaluation of endometrial biopsies obtained from a separate set of mares (n = 3) on Day 6 post ovulation, both before and after uterine lavage, revealed no difference in the accumulation of inflammatory cells, suggesting adverse effects of lavage on fertility may have been due to excessive removal of spermatozoa from the uterus during the lavage process or damage to oviductal spermatozoa.

Effects of postparturient uterine lavage on uterine involution in the mare.

Eighteen postparturient mares were used to evaluate effects of uterine lavage on uterine involution. Mares were randomly assigned to one of three treatment groups: Group 1 (seven mares), no lavage; Group 2 (five mares), lavage on Day 3 post partum; and Group 3 (six mares), lavage on Days 3, 4, and 5 post partum. Five liters sterile physiologic saline, prewarmed to 42 degrees C, were used for each lavage. Transrectal ultrasound examination of the reproductive tract was performed on Day 11 post partum to detect the presence of free fluid in the uterine lumen, to estimate the cross-sectional diameter of the uterine horns and body, and to determine if ovulation had occurred. Endometrial biopsies were also taken on Day 11 post partum to evaluate endometrial histologic characteristics. Lavage had no effect (P>0.05) on diameter of the uterine body or previously gravid uterine horn, presence of fluid in the uterine lumen, or number of mares which had ovulated by Day 11 post partum. Histologic characteristics of the endometrium (height of luminal epithelium, gland depth, relative gland vclume, and inflammatory-cell score) were not affected by treatment (P>0.05). Postpartum uterine lavage did not significantly affect uterine involution by the parameters measured in normal-foaling mares at Day 11 post partum.

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen.

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25 x 10(6) spermatozoa/ml with EZ-Mixin CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25 x 10(6) spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at -20 degrees C until warmed to 37 degrees C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated (r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.

Regulation of estrus and ovulation in mares with progesterone or progesterone and estradiol biodegradable microspheres with or without PGF2alpha.

A single injection of a microsphere preparation, designed to deliver 1.25 gm progesterone and 100 mg estradiol-17beta at a controlled rate, for a duration of 12 to 14 days, produces accurate control of estrus and fertile ovulations in mares. Treatment is followed by PGF2alpha injection 14 days after steroid injection. The objectives of the present study were to determine whether estradiol added to the progesterone treatment or PGF2alpha administered at the end of the steroid treatment regimen, would improve synchronization of estrus and ovulation. A total of 45 cyclic horse mares was randomly assigned to 1 of 5 treatment groups as follows: Group 1 (control, n=9) sterile microsphere vehicle+sterile PGF2alpha vehicle 14 days after treatment with microsphere vehicle; Group 2 (n=9) progesterone and estradiol microspheres+PGF2alpha 14 days after treatment with microspheres; Group 3 (n=9) progesterone and estradiol microspheres+PGF2alpha vehicle 14 days after treatment with microspheres; Group 4 (n=9) progesterone + PGF2alpha 14 days after treatment with microspheres; and Group 5 (n=9) progesterone+PGF2alpha vehicle 14 days after treatment with microspheres. Addition of estradiol (P<0.05) or PGF2alpha (P<0.05) to the treatment regimen increased synchronization efficary by reducing variation in days to ovulation. All treatments significantly reduced variation in days to estrus compared with that of the controls; however, mares in the progesterone groups had an increased incidence of silent or shortened estrous behavior (2 days in duration compared with mares treated with progesterone (78 vs 33%, respectively). Therefore, estradiol and PGF2alpha each appear to reduce variation in days to ovulation while estradiol seems to promote better expression of posttreatment estrous behavior.