Effects of seminal plasma and flash-freezing on DNA structure of stallion epididymal sperm exposed to different potentiators of DNA damage.

The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP+) or unexposed (SP-) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO4) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP- vs. SP+) or freezing treatment (FS vs. FZ; non-significant) on measures of any DNA assays (i.e., 8-hydroxy, 2'deoxyguanosine [8OHdG], TUNEL, or sperm chromatin structure [SCSA] assays). Group FZ was more susceptible than Group FS to potentiators of DNA damage. Percent 8OHdG-positive sperm was higher in Group FZ/SP- treated with FeSO4 than all other groups (P < 0.05). Percent TUNEL-positive sperm was similar among FZ/SP- groups treated with DTT, FeSO4, or DNase (non-significant) and was higher in these groups than all other treatments (P < 0.05). Percent COMP-αt was higher following treatment with DNase or DTT, as compared to their respective controls, regardless of prior exposure to SP (P < 0.05). Overall, sperm DNA structure was unaffected by seminal plasma or freezing treatment when samples were not exposed to potentiators of sperm DNA damage; however, marked differences were identified in DNA structure when sperm were challenged with chemical, oxidative or enzymatic treatments. These results highlight the importance of challenging DNA structure prior to analysis. The use of potentiators of DNA damage provided a model to evaluate sperm DNA structure following exposure of sperm to various experimental treatments.

The effects of antibiotic type and extender storage method on sperm quality and antibacterial effectiveness in fresh and cooled-stored stallion semen.

Two experiments were conducted to evaluate the effects of antibiotic-containing extender of on sperm quality and control of bacterial growth. In Experiment 1, ejaculates were diluted in extender containing no antibiotics, potassium penicillin G-amikacin disulfate (PEN-AMIK), ticarcillin disodium-potassium clavulanate (TICAR-CLAV), piperacillin sodium/tazobactam sodium (PIP-TAZ), or meropenem (MERO). In freshly extended semen, only slight differences were detected among some antibiotic treatments for total sperm motility, curvilinear velocity, and viable acrosome-intact sperm (P < 0.05). In cool-stored semen, slight differences were also detected among certain antibiotic treatments for curvilinear velocity and chromatin integrity (P < 0.05). In Experiment 2, ejaculates were diluted in extender and subjected to no bacterial spiking, or inoculated with lower or higher doses of K. pneumoniae or P. aeruginosa. Following cooled storage of semen, colony forming units/ml (CFU/mL) were less in PEN-AMIK (706 ±â€¯244) and MERO (1576 ±â€¯1076) treatment groups than in TICAR-CLAV (4678 ±â€¯1388) or PIP-TAZ (8108 ±â€¯3198) treatment groups (P < 0.05). The CFU/mL were lower in all antibiotic-containing treatment groups than the control group (18478 ±â€¯4374; P < 0.05). The percentage of culture plates containing no bacterial growth in unspiked semen was greater in PEN-AMIK (75%) than PIP-TAZ (15%) or TICAR-CLAV (20%; P < 0.05). The percentages of culture plates containing no bacterial growth in semen spiked with a lower doses of K. pneumoniae or P. aeruginosa were higher in PEN-AMIK (70% and 50%, respectively) then in all other treatment groups (0-40% and 0-15% for K. pneumonia and P. aeruginosa, respectively; P < 0.05); however, complete control of bacterial load was only modest even with PEN-AMIK. In both experiments, freezing and thawing extender prior to use did not have any appreciable detrimental effect on sperm quality or antibiotic efficacy. In summary, all antibiotics tested had minimal effects on measures of sperm quality in fresh or cool-stored semen extenders; however, PEN-AMIK, followed by MERO, yielded the best results in terms of antimicrobial efficacy. None of the antibiotic types controlled bacterial growth, in comparison with the antibiotic-free control group, when extended semen was spiked with a high concentration of Pseudomonas aeruginosa. Cooled storage of extended semen reduced bacterial growth in comparison with freshly extended semen.

The effect of flash-freezing temperature on stallion sperm DNA structure.

The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabilities were determined. All comet tail measures were higher following any flash-freezing method, as compared to fresh sperm DNA (P < 0.05), with no difference among flash-frozen treatments (P > 0.05). For most comet variables, intra- and inter-assay variabilities were <10%. Intra- and inter-stallion variabilities revealed that comet head length (HL) and width (CW) were less variable as compared to comet tail values, i.e., % comet tail DNA (T-DNA), tail length (TL), tail moment (OTM), and tail migration (TM). Certain comet tail values in fresh (% T-DNA, and OTM) and flash-frozen sperm (OTM, % T-DNA, TL, and TM) were correlated to the Sperm Chromatin Structure Assay (SCSA) variable, COMP-αt. The comet tail measures were negatively correlated to % morphologically normal sperm (P < 0.05) and positively correlated to % abnormal heads and premature germ cells (P < 0.05). Variables COMP-αt and % total sperm motility were not correlated to any morphologic sperm feature in this group of stallions (P > 0.05). While significant differences in the structure of the sperm DNA were identified in the flash-frozen as compared to the fresh sperm DNA with the neutral comet assay, it cannot be assumed that these changes are fertility limiting.

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.

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.

Sperm DNA quality evaluated by comet assay and sperm chromatin structure assay in stallions after unilateral orchiectomy.

Unilateral orchiectomy (UO) may interfere with thermoregulation of the remaining testis caused by inflammation surrounding the incision site, thus altering normal spermatogenesis and consequently sperm quality. Two measures of sperm DNA quality (neutral comet assay and the sperm chromatin structure assay [SCSA]) were compared before UO (0 days) and at 14, 30, and 60 days after UO to determine whether sperm DNA changed after a mild testis stress (i.e., UO). The percent DNA in the comet tail was higher at 14 and 60 days compared to 0 days (P < 0.05) after UO. All other comet tail measures (i.e., length, moment, migration) were higher at all time periods after UO compared to 0 days (P < 0.05). Two SCSA measures (mean-αt, mode-αt) increased at 14 days after UO (P < 0.05), whereas two measures (SD-αt and COMP-αt) did not change. This study identified a decrease in sperm DNA quality using both the neutral comet assay and the SCSA, which was not identified using traditional measures of sperm quality.

The relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses.

The relationship between the quality of cool-shipped stallion semen and fertility has not been adequately described. This study evaluated sperm quality of cool-shipped semen from 459 ejaculates (N = 130 stallions) that were used for insemination of 196 embryo donor mares (n = 496 estrous cycles). Embryo recovery rate (ERR; %) increased, as all sperm measures (e.g., motility, viability, DNA quality, morphology, concentration, and total number) increased. Threshold values are reported for each sperm quality measure (e.g., total sperm motility ≥ 65%) that separate two ERR groups (e.g., average: ∼50% ERR; high: ∼65% ERR).

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.

Determination of daily sperm production in stallion testes by enumeration of germ cells in homogenates.

Thirty adult stallion testes were selected with high (n = 15) and low (n = 15) Daily Sperm Production (DSP)/testis. Parenchymal samples were prepared for morphometric analysis, and the numbers of germ cells and Sertoli cells were determined. Testicular samples were homogenized, and germ cells and Sertoli cells were enumerated using phase contrast microscopy. Numbers of germ cells and Sertoli cells and potential DSP during spermatogenesis were determined. Significant correlations existed between morphometric and homogenate determinations of number per testis of preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.58; P < 0.001), pachytene plus diplotene primary spermatocytes (r = 0.67; P < 0.0001), all primary spermatocytes (r = 0.67; P < 0.0001), round spermatids (r = 0.72; P < 0.0001), and Sertoli cells (r = 0.70; P < 0.0001). Significant correlations (P < 0.0001) existed between morphometric and homogenate determination of DSP/testis based on preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.78), pachytene plus diplotene primary spermatocytes (r = 0.88), and round spermatids (r = 0.85). Using morphometric determination as the standard, the sensitivity (i.e., ability to detect low DSP/testis) and specificity (i.e., ability to detect high DSP/testis) by homogenate enumeration of germ cells was 81 and 93% for round spermatids, 100 and 24% for pachytene plus diplotene primary spermatocytes, and 67 and 87% for preleptotene, leptotene plus zygotene primary spermatocytes, respectively. Enumeration of primary spermatocytes in homogenates was less accurate than enumeration of round or elongated spermatids. Enumeration of round and elongated spermatids in homogenates was a rapid and useful method for determining DSP in horses, and it may prove to be a useful technique for quantitating potential DSP from testicular biopsies.

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production.

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.

The effects of EDTA-Tris infusion on the equine endometrium.

Four groups of five pony mares each were used to determine if the intrauterine infusion of EDTA-Tris solution caused adverse effects on the endometrium. The uteri of mares were infused with either saline or EDTA-Tris solution or biopsied or sham-biopsied without infusion. Acute endometritis developed in one (20%) to three (60%) mares in each group during the seven days following treatment, but there were no differences (P > 0.05) in the incidence of endometritis among the groups. Endometrial fibrosis was not evident in biopsies taken on days 14, 30 and 60 following infusion of saline or EDTA-Tris. It was concluded that the endometrial response to saline and EDTA-Tris was not different and that EDTA-Tris may be a useful adjunct to treatment of uterine infections in the mare.

Comparison of three containers used for the transport of cooled stallion semen.

Three containers commonly used to transport cooled equine semen (Equitainer, ExpectaFoal and a Swedish-designed semen-transport container, previously called the Salsbro Box and now called Equine Express) were compared, using four ejaculates from each of three stallions. Each ejaculate was diluted to a spermatozoal concentration of 25 x 10(6)/ml with a nonfat dry milk-glucose extender containing amikacin sulfate (1 mg/ml) and potassium penicillin G (1000 units/ml). Extended semen was divided into three 40-ml aliquots for placement in each of the three semen-transport containers. The extended semen was stored in the containers for 24 h prior to analysis. Stored semen was warmed for 15 min at 37 degrees C, then video records of sperm motility were obtained for evaluation using a Hamilton-Thorne motility analyzer equipped with a stage warmer set at 37 degrees C. The temperature of 40-ml aliquots of semen extender stored in each container was also measured for 60 h using a copper-constantan thermocouple placed in the center of the stored samples. Intervals from onset of storage until sample temperature exceeded 10 degrees C during the warming phase were 27.5, 33.5 and 53 h, for the Expecta-Foal, Equine Express and Equitainer, respectively. Semen extender stored in the Equitainer compared most favorably to ideal cooling rates and storage temperatures published previously. Following a 24-h storage period, the mean percentages of motile, progressively motile, and rapidly motile spermatozoa, as well as the mean spermatozoal curvilinear velocity were similar (P > 0.05) among the three containers.

Factors affecting spermatogenesis in the stallion.

Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. Seminiferous tubules are composed of somatic cells (myoid cells and Sertoli cells) and germ cells (spermatogonia, spermatocytes, and spermatids). Activities of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytogenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number to haploid and yield four spermatids. Spermiogenesis is the differentiation without division of spherical spermatids into mature spermatids which are released from the luminal free surface as spermatozoa. The spermatogenic cycle (12.2 days in the horse) is superimposed on the three major divisions of spermatogenesis which takes 57 days. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis, and quantitative measures are related to number of spermatozoa in the ejaculate. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest seasonal impact on horses occurs during spermatocytogenesis. Daily spermatozoan production is related to the amount of germ cell degeneration, pubertal development, season of the year, and aging. Number of Sertoli cells and amount of smooth endoplasmic reticulum of Leydig cells and Leydig cell number are related to spermatozoan production. Seminiferous epithelium is sensitive to elevated temperature, dietary deficiencies, androgenic drugs (anabolic steroids), metals (cadmium and lead), x-ray exposure, dioxin, alcohol, and infectious diseases. However, these different factors may elicit the same temporary or permanent response in that degenerating germ cells become more common, multinucleate giant germ cells form by coalescence of spermatocytes or spermatids, the ratio of germ cells to Sertoli cells is reduced, and spermatozoan production is adversely affected. In short, spermatogenesis involves both mitotic and meiotic cell divisions and an unsurpassed example of cell differentiation in the production of the spermatozoon. Several extrinsic factors can influence spermatogenesis to cause a similar degenerative response of the seminiferous epithelium and reduce fertility of stallions.

Estrous cycle characteristics and response to estrus synchronization in mammoth asses (Equus asinus americanus).

Breeding records from a herd of mammoth asses (Equus asinus americanus) maintained on pasture in southeast Texas from 1990 to 1998 were reviewed. Jennies were pasture or hand mated, and estrus was either observed while the jennies were on pasture or when exposed to a jack after being penned. Eighty-one estrus periods and 43 diestrus intervals were recorded in 33 jennies over 4 seasons of the year (January-March, April-June, July-September, and October-December). Estrous cycle length and the duration of estrus were similar among seasons. Over all seasons, estrous cycle length was 23.3 +/- 2.6 d, duration of estrus was 5.9 +/- 2.1 d, and diestrus length was 17.4 +/- 2.6 d (mean +/- SD). During these same 9 yr, 58 injections of PGF2 alpha (5 mg, i.m.) were administered to 38 jennies without regard to stage of estrous cycle. Seventy-six percent (44/58) of the jennies showed signs of estrus after PGF2 alpha treatment, with an interval to estrus of 4.4 +/- 1.6 d and a duration of estrus of 5.6 +/- 1.7 d. Two estrus synchronization schemes were also assessed. Trial 1 was performed in October to November 1996, and Trial 2 was performed in February to March 1998. In Trial 1 (Group PE + PGF, n = 10), each jenny was injected intramuscularly once daily for 10 d with 150 mg progesterone and 10 mg estradiol-17 beta in sesame oil, and PGF2 alpha (10 mg) was injected intramuscularly on the last day of treatment. In Trial 2 (Group PGF-2X, n = 11), each jenny was injected intramuscularly twice, 16 d apart, with 10 mg PGF2 alpha. All Group PE + PGF jennies responded to treatment. One jenny in Group PGF-2X did not respond to either injection of PGF2 alpha, while 2 jennies responded to the first but not the second PGF2 alpha injection (8 of 11 jennies returned to estrus and ovulated after the second PGF2 alpha injection). Duration of estrus was 6.8 +/- 1.9 d for Group PE + PGF and 7.1 +/- 1.8 d for Group PGF-2X jennies. Interval to estrus and interval to ovulation following the last treatment were 9.0 +/- 0.9 d and 14.5 +/- 1.7 d, respectively, in Group PE + PGF jennies, and 4.5 +/- 0.9 d and 10.4 +/- 1.8 d, respectively, for Group PGF-2X jennies. In summary, estrous cycle characteristics of mammoth asses are similar to those reported for standard jennies, and estrus synchronization schemes used in horses are effective in mammoth asses.

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 cooling rate and storage temperature on equine spermatozoal motility parameters.

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.

Investigation of the representativeness of a single endometrial sample and the use of trichrome staining to aid in the detection of endometrial fibrosis in the mare.

The uteri of five mares were removed and endometrial samples were procured from 12 specific locations in the uteri and the samples were processed and duplicate sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome stains. The samples were interpreted in a blind manner by one person, and pathologic changes were classified according to Kenney (1). An assessment of stromal fibrous connective tissue and focal periglandular fibrosis or fibrotic nests was made. There were no significant differences in luminal epithelial cell heights or in the occurrence and severity of stromal fibrous connective tissue, focal periglandular fibrosis, or lymphatic lacunae among locations (P > 0.05). There was an effect of location on the occurrence and severity of inflammation (P < 0.05). If only inflammation was considered in categorization, this would have resulted in changing the category in 9 of 60 samples. There was no increase in tendency for inflammation, fibrosis or lymphatic lacunae to occur in the horns versus the body of the uterus, nor of the dorsal versus the ventral uterus (P > 0.05). There was no effect (P > 0.05) of type of stain on the ability to detect incidence and severity of focal periglandular fibrosis. There was an effect (P < 0.05) of type of stain on the ability to detect the incidence and severity of stromal fibrous connective tissue. The use of the trichrome stain showed slightly increased distribution of stromal fibrous connective tissue.