B.A. Ball

Pregnancy rates at Days 2 and 14 and estimated embryonic loss rates prior to day 14 in normal and subfertile mares

Pregnancy rates at Days 2 and 14 postovulation were determined for 15 normal mares and 15 subfertile mares. Embryonic loss rates were estimated by the difference in the Day 2 and Day 14 pregnancy rates. Mares were artificially inseminated with the pooled ejaculates from three stallions, and the embryonic vesicle was detected with ultrasonography at Days 9, 10, 12 and 14. Mares were short-cycled with prostaglandin F(2) alpha (PGF(2alpha)) and rebred to the same stallions, and the Day 2 pregnancy rates were determined by recovery of cleaved ova (embryos) from the surgically excised oviducts. Significantly more (P < 0.01) normal versus subfertile mares were pregnant at Day 14 (12 15 vs 3 15 ). There was no significant difference in the Day 2 pregnancy rate for normal versus subfertile mares (10 14 vs 11 14 ). There were no significant differences (P > 0.5) in the mean number of blastomeres per embryo or in the mean diameter of embryos recovered at Day 2 from normal or subfertile mares. The estimated embryonic loss rate was significantly lower (P < 0.01) for normal verusus subfertile mares (0 10 vs 8 11 ). Fertilization rates were similar for normal and subfertile mares; however, subfertile mares had a higher embryonic loss rate prior to Day 14 postovulation.

Use of a low-volume uterine flush for microbiologic and cytologic examination of the mare's endometrium

Abstract A guarded uterine swab and a low-volume uterine flush were compared for microbiologic and cytologic examination of the endometrium of young, normal mares (n = 24) and aged, subfertile mares (n = 27). Mares from each group had clitoral sinus swabs, guarded endometrial swabs (X2), uterine flushes and endometrial biopsy samples taken for microbiologic, cytologic and histologic examination. The guarded swab technique yielded significantly fewer (P 0 24 vs 6 24 ) and subfertile mares ( 4 27 vs 14 27 ). The swab technique also yielded significantly fewer (P 12 27 vs 20 27 ); however, there was no significant difference (P > 0.1) for the two techniques for cytologic results in normal mares ( 3 24 vs 7 24 ). Ureaplasma spp. were isolated from the uterine flushes ( 3 27 ) and clitoral sinus swabs ( 6 27 ) from subfertile mares. Mycoplasma spp. were isolated from the uterine flush from one subfertile mare and clitoral sinus swab from another subfertile mare. Significantly fewer (P 5 24 vs 25 27 ). Based on microbiologic and cytologic results, the low-volume uterine flush appeared superior to the guarded endometrial swab for the diagnosis of endometritis in mares.

Morphometry of stallion spermatozoa by computer-assisted image analysis

Metric measurements of stallion spermatozoal heads were determined for live, unfixed spermatozoa and for Feulgen-stained spermatozoa by videomicroscopy and computerized image analysis. Two ejaculates were collected from each of five stallions of normal fertility. Air-dried semen smears were Feulgen-stained, and live, unfixed spermatozoa were examined as wet-mount preparations. For Feulgen-stained spermatozoa, videoimages (x3850) were captured, and sperm heads were detected via image segmentation and particle analysis. For live, unfixed spermatozoa, phase contrast videoimages (x3850) were measured to determine width and length of the sperm head. For Feulgen-stained spermatozoa, there were significant effects (P < 0.001) of stallion and ejaculate on measured parameters of area, circumference, and the length and width of the sperm head. For live, unfixed spermatozoa, there were significant effects of stallion on length and width and of ejaculate on length of the sperm heads. There was a very poor correlation between length and width of sperm heads between Feulgen-stained and live, unfixed spermatozoa. Two indices of sperm shape (oval factor and aspect ratio) were also determined. Both aspect ratio and oval factor were significantly affected by stallion (P < 0.001); however, oval factor was not affected by ejaculate and therefore may represent a less variable determination of sperm head shape across stallions. Overall, length and width of stallion sperm heads were larger (P < 0.01) for live, unfixed spermatozoa than for Feulgen-stained spermatozoa (length: 6.3 +/- 0.4 vs 5.08 +/- 0.44; width: 3.08 +/- 0.34 vs 2.71 +/- 0.28 mum, respectively). Computerized image analysis may be useful as a means to objectively measure sperm head dimensions in the stallion and could be useful in future studies to determine associations with stallion fertility.

THE USE OF PENTOXIFYLLINE TO IMPROVE MOTILITY OF CRYOPRESERVED EQUINE SPERMATOZOA

Pentoxifylline was evaluated as a method to increase motility of cryopreserved equine spermatozoa. In a preliminary experiment, pentoxifylline (3.5 mM or 7.0 mM) was added to extended semen that was chilled to 4 degrees C. Motility was evaluated at 8-h intervals for 48 h. The addition of 3.5 or 7.0 mM pentoxifylline appeared to increase the motility of chilled spermatozoa compared to controls. Based on these results, similar concentrations of pentoxifylline were added to semen either before or after cryopreservation. The addition of pentoxifylline (3.5 or 7.0 mM) to semen before cryopreservation significantly (P < 0.001) decreased total and progressive motility compared to controls. However, the addition of pentoxifylline (3.5 or 7.0 mM) to cryopreserved semen immediately after thawing significantly (P < 0.01) increased total and progressive motility compared to controls. These results indicate that pentoxifylline enhanced the postthaw motility of cryopreserved equine semen when added after thawing. Further research is required to evaluate the effect of pentoxifylline on the fertility of cryopreserved equine semen.

In vitro development of Day-2 equine embryos co-cultured with oviductal explants or trophoblastic vesicles

This study compared the in vitro development of Day-2 equine embryos co-cultured with either trophoblastic vesicles or oviductal explants. Embryos were collected surgically from the oviducts of pony mares 2 d after ovulation and assessed for stage of development. Culture medium was Hams F12 and Dulbeccos Modified Eagles Medium (50:50 v/v) in a humidified atmosphere of 5% CO(2) in air at 38.5 degrees C with either trophoblastic vesicles or oviductal explants. The quality score of embryos was assessed daily. After 4 d in culture, embryos were stained (Hoechst 33342) and evaluated with epifluorescence to determine the number of nuclei present. Six of seven embryos co-cultured with oviductal examples developed to the morula/blastocyst stage, while four of seven embryos co-cultured with trophoblastic vesicles developed to the morula stage. More (P = 0.1) embryos co-cultured with oviductal explants reached the blastocyst stage than embryos co-cultured with trophoblastic vesicles (3/7 vs 0/7, respectively). The number of cells was higher (P = 0.1) for embryos co-cultured with oviductal explants than for embryos co-cultured with trophoblastic vesicles (162.6 +/- 32 vs 87.3 +/- 28, respectively). The number of cells for embryos co-cultured with either oviductal explants or trophoblastic vesicles appeared to be lower than for embryos matured in vivo that were recovered from the uterus at Day 6 (378, 399, >1000). The co-culture of early equine embryos in a completely defined medium with either trophoblastic vesicles or oviductal explants can support development to at least the morula stage. The co-culture of embryos with oviductal explants resulted in superior development of four-to eight-cell embryos, as indicated by the proportion that reached the blastocyst stage and by the number of cells.

Survival of equine embryos transferred to normal and subfertile mares

To test the hypothesis that an abnormal uterine environment was a cause of early embryonic loss in subfertile mares, morphologically normal embryos were transferred to normal mares (n = 20) and subfertile mares (n = 20), and embryo survival rates were compared. Embryos were recovered nonsurgically at Days 7 to 8 postovulation and transferred surgically to normal and subfertile mares that had ovulated on the same day or within 2 d after a donor. Survival of transferred embryos was monitored by ultrasonography of the recipient mares uterus from Day 9 through Day 28 postovulation. There were no significant differences (P > 0.5) in the embryo survival rates at Day 12 (11 20 vs 9 20 ) or Day 28 (10 20 vs 8 20 ) for normal or subfertile mares, respectively. The uterine environment of subfertile mares was apparently adequate to support the development of transferred embryos from Days 7 or 8 through Day 28 postovulation.

Survival of equine embryos co-cultured with equine oviductal epithelium from the four- to eight-cell to the blastocyst stage after transfer to synchronous recipient mares

In this study we examined the ability of equine oviductal epithelial cells (OEC) to support the development of four- to eight-cell equine embryos in vitro and investigated the ability of co-cultured embryos to continue normal development after transfer to synchronous recipient mares. Equine embryos obtained at Day 2 after ovulation were cultured with or without OEC for 5 days. Those OEC co-cultured embryos that reached the blastocyst stage and embryos recovered from the uterus at Day 7 were surgically transferred to synchronous recipient mares. Co-culture with OEC improved (P < 0.01) development of four- to eight-cell embryos to blastocysts compared to medium alone (11/15 vs 0/6) during 5 days in vitro. Embryos co-cultured with OEC were smaller (P < 0.05) and more delayed in development than Day-7 uterine blastocysts. There was no difference in the Day-30 survival rate of co-cultured blastocysts (3/8) or Day-7 uterine blastocysts (5/8) after transfer to recipient mares. These results indicate that co-culture with OEC can support development of four- to eight-cell equine embryos in vitro and that co-cultured embryos can continue normal development after transfer to recipient mares.

USE OF PREGNANCY-SPECIFIC PROTEIN-B AND ESTRONE SULFATE FOR DETERMINATION OF PREGNANCY ON DAY 49 IN FALLOW DEER (DAMA DAMA)

The objective of this study was to determine if pregnancy specific protein-B (PSPB) and estrone sulfate (E(1)SO(4)) could be used to determine pregnancy status in fallow deer (Dama dama ). Forty mature does were synchronized for estrus with an intravaginal progesterone-releasing device (CIDR) and then artificially inseminated via laparoscopy with frozen semen on one day. Ultrasound examination and jugular blood sampling were done 49 days later. Transrectal ultrasonography was done to presumptively determine the pregnancy status at the time of blood sampling. Serum estrone sulfate concentrations were significantly (P < 0.05) greater in pregnant (n=31) than nonpregnant (n=9) females at 49 days of gestation (166.7 +/- 25.9 pg/ml vs 36.3 +/- 11.1 pg/ml, respectively). The percentage of [(125)I]PSPB bound was significantly (P < 0.01) lower when sera of pregnant (n=29) versus nonpregnant (n=9) females was added to RIA tubes (63.7 +/- 1.6% vs 98.1 +/- 1.6%, respectively). There were 30 fawns born from the group of females that were diagnosed pregnant based on ultrasound. We conclude that estrone sulfate and PSPB can be used to determine pregnancy status in fallow deer at 49 days of gestation.

Culture of equine trophoblastic vesicles in vitro

Trophoblastic vesicles have been used to study early embryonic development and maternal recognition of pregnancy in domestic animals. The purpose of this study was to characterize the formation of trophoblastic vesicles from Day-12 to Day-16 equine conceptuses. Conceptuses (n = 19) were collected nonsurgically from mares, the capsule was removed, and the conceptus (trophoblast and inner cell mass) was dissected into 2- to 4-mm fragments. Conceptus fragments were cultured in either Hams F10 (HF10) or Minimum Essential Media (MEM) with 10% fetal bovine serum (FBS) in 24-well plates. Plates were incubated at 37 degrees C in a humidified atmosphere of 90% N(2), 5% O(2), and 5% CO(2) and were examined at 48 and 96 h for the number and diameter of trophoblastic vesicles formed. There was no significant difference (P > 0.1) between HF10 and MEM in the diameter of trophoblastic vesicles at 48 or 96 h of culture. There was, however, a significant increase (P < 0.01) in the number of trophoblastic vesicles per conceptus between 48 and 96 h of culture for HF10 and MEM. The mean diameters of trophoblastic vesicles after 96 h in culture were 396.4 +/- 19.9 mum and 415.0 +/- 12.0 mum for HF10 and MEM, respectively. Histologic sections of trophoblastic vesicles revealed a bilaminar structure consistent with the presence of trophectoderm and endoderm. Squash preparations of trophoblastic vesicles contained cells similar to those recognized in squash preparations of fresh conceptuses. Areas of increased cell density that resembled the inner-cell mass were seen in both fresh trophoblastic vesicles and in sections of trophoblastic vesicles. Equine trophoblastic vesicles may be useful to further the study of early embryonic development and pregnancy recognition in mares.

Influence of exogenous progesterone on early embryonic development in the mare

The influence of exogenous progesterone on the development of equine oviductal embryos was determined based upon the recovery of Day-7 uterine blastocysts from treated mares (n=13) that were given 450 mg progesterone daily between Days 0 and 6 and from untreated control mares (n=13). Daily administration of 450 mg progesterone in oil significantly (P<0.02) increased serum progesterone concentrations in the treated mares. There was no significant difference in the recovery rate of Day-7 embryos between treated and control mares (8/13 versus 6/13, respectively). Embryonic development, assessed by morphologic evaluation, embryo diameter, and number of cell nuclei was not significantly different for embryos from treated and from control mares. The results of this study indicate that administration of progesterone beginning on the day of ovulation does not affect the embryo recovery rate or embryonic development, based on evaluation of uterine blastocysts recovered at Day 7 after ovulation.