The effects of cooling and vitrification of embryos from mares treated with equine follicle-stimulating hormone on pregnancy rates after nonsurgical transfer

Equine embryos can remain viable for 12 to 24 hours when cooled and stored at 5°C.¹ Cryopreservation of embryos would allow for long-term preservation of genetic material and more efficient management of embryo recipients. This study compared pregnancy rates after transfer of equine embryos vitrified within 1 hour of collection or cooled for 12 to 19 hours before vitrification. Mares (N = 40) were superovulated using equine follicle-stimulating hormone (eFSH). Embryos were recovered 6.5 days after ovulation or 8 days after human chorionic gonadotropin. Forty morulae or early blastocysts with a grade of 1 to 2 and <300 mm in diameter were randomly assigned to 1 of 2 treatments: Group 1 (n = 20), washed 4 times in a commercial holding medium and then vitrified; Group 2 (n = 20), washed 3 times and then stored in the same holding medium at 5°C to 8°C in a passive cooling device for 12 to 19 hours before being vitrified. To thaw, embryos were warmed by holding the straw in air at room temperature for 10 seconds and then submerged in a water bath (20°C to 22°C) for an additional 10 seconds. The contents of the straw were transferred directly into a recipient that had ovulated 4 to 6 days previously. There were no differences (P > .05) in embryo diameter, grade, or morphology score between treatment groups before vitrification. Pregnancy rates (day 16) were not different (P > .05) between embryos vitrified immediately after collection (15 of 20; 75%) and embryos cooled for 12 to 19 hours before vitrification (13 of 20; 65%). Based on these results, small equine embryos (<300 mm) can be stored at 5°C to 8°C for 12 to 19 hours before vitrification without a significant loss of viability.     

Exogenous eFSH, follicle coasting, and hCG as a novel superovulation regimen in mares

The objective of this study was to evaluate various equine follicle-stimulating hormone (eFSH) treatment protocols and the effect of “follicle coasting” on ovulation and embryo recovery rates in mares. Cycling mares (n = 40) were randomly assigned to one of four groups 7 days after ovulation: (1) 12.5 mg eFSH twice daily until follicles were 35 mm or larger; (2) 12.5 mg eFSH twice daily until follicles were 32 mm or larger; (3) 12.5 mg eFSH twice daily for 3.5 days followed by 12.5 mg eFSH enriched with luteinizing hormone (LH) twice daily until follicles were 35 mm or larger; and (4) 25 mg eFSH once daily until follicles were 32 mm or larger. Mares in groups 1 and 3 were injected with human chorionic gonadotropin (hCG) (2500 IU intravenously) at the end of eFSH treatment, whereas mares in groups 2 and 4 were given hCG approximately 42 and 54 hours, respectively, after the last eFSH treatment (“follicle coasting”). Nonsurgical embryo collection was performed 6.5 to 7.5 days after ovulation. Each mare experienced a nontreated estrous cycle before being reassigned to a second treatment. Ovulation rates for mares in treatment groups 1 to 4 were 3.3 ± 0.4, 4.1 ± 0.4, 3.5 ± 0.4, and 2.8 ± 0.4 (mean ± SEM; P < .05), respectively. One or more embryos were recovered from more than 80% of mares in each treatment group, and embryo recovery rate per flush was similar among treatment groups (1.9 ± 0.3, 2.6 ± 0.3, 1.9 ± 0.3 and 1.9 ± 0.3, respectively; P > .05). The overall embryo recovery rate was 2.1 ± 1.5 embryos per flush. In summary, ovulation rate was higher for mares treated with eFSH (3.4 ± 0.4) compared with non-treated controls (1.1 ± 0.2). Ovulation rate in mares in which hCG was delayed (follicle coasting) was higher (P < .05) when treatments were given twice per day versus once per day. Administration of equine luteinizing hormone (eLH) in conjunction with eFSH did not have an advantage over mares treated only with eFSH.     

Effects of transportation on early embryonic death in mares

Incidence of early embryonic death (EED) and associated changes in serum cortisol, progesterone and plasma ascorbic acid (AA) in transported mares were investigated. Mares were transported for 472 km (9 h) during either d 16 to 22 (T-3 wk, n = 15) or d 32 to 38 (T-5 wk, n = 15) of gestation. Blood samples were drawn from control, nontransported mares (NT-3 wk, NT-5 wk, n = 24) and transported mares pre-trip, midtrip, and at 0, 12, 24, 48 and 72 h post-transport and daily for the next 2 wk. Incidence of EED between transported and nontransported mares was not different (P greater than .05). Serum cortisol in all transported mares increased (P less than .05) relative to pre-trip values at midtrip and 0 h post-transport. Relative to NT mares, serum cortisol was higher (P less than .05) at midtrip in T-3 wk mares and 0 h post-transport in T-5 wk mares. Serum progesterone in all T mares increased (P less than .05) at midtrip relative to pre-trip values and was higher (P less than .05) in T-3 wk mares than in NT-3 wk mares at midtrip and 0 h post-transport. Post-transport decreases (P less than .05) in concentrations of progesterone were observed in mares that aborted. Plasma AA in transported mares increased (P less than .05) at midtrip in T-5 wk mares and decreased (P less than .05) relative to pre-trip values at 24 and 48 h post-transport (T-3 wk and T-5 wk mares, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fetal mummification in a mare

A single mummified fetus was removed from the uterus of a 23-year-old mare that had been bred approximately 30 months previously. The mare had received supplemental progestin therapy for approximately 150 days after ovulation. This case represents the longest recorded occurrence of fetal mummification in the mare. Progestion administration may have contributed to the initial retention of the fetus in the uterus.     

Immunohistochemical Expression of Growth Factors in the Follicular Wall of Normal and Cystic Ovaries of Sows

The expression of growth factors was evaluated immunohistochemically in normal and cystic ovaries of sows. The immunohistochemically stained area (IHCSA) was quantified by image analysis to analyse the expression of these proteins in the follicular wall of secondary, tertiary and cystic follicles. IGF‐I immunoreactivity was strong in the granulosa cell layer (GC), moderate in the theca interna (TI) and mild in the theca externa (TE) of the normal follicles. There was severe reduction of the labelling to IGF‐I in the GC of the follicular and luteinized cysts. In the normal follicles, the reactivity for IGF‐II was very similar to pattern noted in IGF‐I. There was reduction of the IHCSAs in the GC of the follicular and luteinized cysts, but the decrease was not significant. The staining of the IGF‐II in the TI and TE of the cysts was increased, in comparison with normal follicles. The IHCSAs for VEGF were higher in the GC and TE of the normal follicles in contrast to TI, but this difference was noted only in the tertiary follicle. The VEGF reactivity increased in the GC of the cysts, in relation to normal follicles. The results of the current study show that the formation of ovarian cysts in sows is associated with alterations in the immunohistochemical expression of some growth factors.     

Presence of Bacteria on the External Genitalia of Healthy Stallions and its Transmission to the Mare at the Time of Breeding by Live Cover

The current field study used thoroughbred stallions and mares from central Kentucky to investigate the occurrence of potentially pathogenic bacteria on the stallion's external genitalia, based on cultures, and investigated the occurrence of bacteria and type of isolate in the mare's uterus after breeding by live cover to stallions with or without positive bacterial cultures. Fifteen thoroughbred stallions and 206 mares from two central Kentucky thoroughbred farms were used during the 2010 and 2011 breeding seasons. Samples for bacteriological evaluation were taken from the prepuce and postejaculate urethra (n = 201) of stallions. Uterine swabs (n = 264) were collected 12-18 hours postbreeding. For statistical analyses, a chi-squared test was used to test the relationship between stallion culture results and postbreeding uterine culture results, as well as the effect of bacterial types found on the stallion cultures with bacterial types found on the postbreeding uterine cultures. Of stallion cultures, 22.4% were positive for potentially pathogenic bacteria, with Streptococcus equi subspecies zooepidemicus (51.1%) being the most common isolate. Uterine cultures resulted in a 29.2% positive rate for potentially pathogenic bacteria, with S. equi subsp. zooepidemicus (90.9%) being the most common. There was no difference (P > .05) in the occurrence of bacteria or type of isolate found on uterine cultures after breeding stallions with or without positive cultures. In conclusion, potentially pathogenic bacteria found on the stallion's external genitalia did not affect the occurrence and type of bacterial isolate found in the mare's uterus after breeding by live cover.