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SummaryReasons for performing study: Transvaginal ultrasound-guided follicle aspiration (TVA) is performed clinically but there is little information available on complications associated with this procedure. Objectives: It is possible that TVA is associated with damage to the ovary and may induce peritonitis or peritoneal adhesions. This study was conducted to determine the effect of repeated TVA on mare health and ovarian status. Methods: Thirty-two mares were used for oocyte recovery via repeated TVA over a 3 year period; different mares were used each year. In Year 1, ovarian status was monitored in 11 mares by transrectal palpation and ultrasonography. In Year 2, 6 of 11 mares underwent abdominocentesis and were examined by laparoscopy after one TVA and again after multiple TVAs. In Year 3, 10 mares underwent multiple TVAs with either a 15 or a 12 gauge needle and the ovaries were removed for examination. Results: Four hundred and twenty-seven aspiration sessions (390 via TVA and 37 via needle placement through the flank) and 3202 follicle punctures (3161 TVA and 41 flank) were performed. One mare developed an ovarian abscess. Transient rectal bleeding was evident after 16% of TVA sessions. No adhesions were found on laparoscopic or gross examination of ovaries and there were minimal changes on histological evaluation. Conclusions: Follicle aspiration carries a small possibility (<0.5%) of ovarian abscess formation. There is a possibility of rectal abrasion or puncture but little gross or histological damage to the ovary. Potential relevance: These results provide a basis for using prophylactic administration of antibiotics after TVA and for advising mare owners of the rare but potential complications associated with the procedure.

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Placement of sperm deep in the equine uterine horn allows fewer sperm to be inseminated while maintaining acceptable fertility, and has been promoted for use in circumstances when fertility would be expected to be low if standard insemination were used (e.g., semen from a subfertile stallion, or frozen-thawed semen). Two main techniques, transrectally guided (TRG) and hysteroscopic (HYS) insemination, have been developed for this purpose; however, there is some controversy regarding their comparative efficacy. This study was conducted to compare pregnancy rates when mares were inseminated by TRG or HYS, using sperm numbers approaching and under the minimum threshold, resulting in reduced fertility. When 1 × 10(6) sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (10/13, 77%) and TRG (11/15, 73%). Similarly, when 0.5 × 10(6) sperm were inseminated, pregnancy rates were not different (P > 0.10) between techniques HYS (3/15, 20%) and TRG (4/13, 31%). Combined pregnancy rates for the two treatments were 13/28 (46%) for HYS and 15/28 (54%) for TRG (P > 0.10). Pregnancy rates using a subthreshold number of sperm were not significantly affected by a deep-horn insemination technique.

Three ejaculates from each of eight stallions were initially centrifuged in INRA 96 extender and spermatozoal pellets were resuspended in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Extended semen was exposed to a fast pre-freeze cooling rate (FAST -semen immediately subjected to cryopreservation) or a slow pre-freeze cooling rate (SLOW -semen pre-cooled at a controlled rate for 80 minutes prior to cryopreservation). After thawing, semen was diluted in initial freezing medium (FM) or INRA 96 prior to analysis of 9 experimental endpoints: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; µm/sec), average-path velocity (VAP; µm/sec), straight-line velocity (VSL; µm/sec), linearity (LIN; %), intact acrosomal and plasma membranes (AIVIAB; %), intact acrosomal membranes (AI; %), and intact plasma membranes (VIAB; %). Mean values for these endpoints were higher following spermatozoal exposure to a slow pre-freeze cooling period, regardless of freezing extender type (P<0.05). The effects of pre-freeze cooling rate on MOT, AIVIAB, AI, and VIAB were more pronounced in spermatozoa cryopreserved in MF extender, as compared to LE extender. Within treatment groups SLOW and FAST, mean MOT, AIVIAB, AI, and VIAB were higher (P<0.05) for spermatozoa cryopreserved in LE extender, as compared to MF extender.

Whereas small equine embryos may be successfully cryopreserved, pregnancy rates after cryopreservation of large equine blastocysts (>300 μm) are low. This has been attributed to their large diameter, failure of cyoprotectants to diffuse into the blastocoele, and/or impedance from the equine embryonic capsule. Previously, we successfully biopsied the trophoblast of equine in vivo-recovered expanded blastocysts (>300 μm; Choi et al. 2009 Reprod. Fertil. Dev. 21, 166-167). In that study, we observed embryonic growth in vitro after warming of a blastocyst vitrified immediately after biopsy. An efficient vitrification method was recently described for ferret embryos, which, like horse embryos, contain a large amount of lipid. This method utilizes very small (250 μm inner diameter) pipettes (Femtotips, Eppendorf; Sun et al. 2008 Biol. Reprod. 79, 832-840). We utilized this technique, with minor modifications, to vitrify equine embryos. In Study 1, the vitrification procedure was performed on equine in vitro-produced blastocysts (<200 μm diameter). All 11 embryos vitrified with the procedure and subsequently warmed grew in culture, showing an increase in diameter over the first 24 h, followed by protrusion of embryonic tissue through holes in the zona pellucida. In Study 2, 10 in vivo-recovered expanded blastocysts, >300 μm diameter, were biopsied using a Piezo drill, causing blastocoele collapse, and were then immediately vitrified. Three of these vitrified blastocysts (346, 399, and 514 μm in diameter) were warmed and transferred to culture; all 3 expanded within 8 h and subsequently proliferated through holes in the embryonic capsule. Seven vitrified in vivo-recovered blastocysts were warmed at the laboratory, shipped to a transfer facility, and transferred transcervically to recipient mares. Five out of 7 transferred blastocysts (71%) resulted in pregnancy at Day 14. Diameters of these embryos before biopsy and vitrification were 360, 398, 415, 434, and 599 μm. The 2 biopsied and vitrified embryos that did not result in pregnancy after transfer were originally 376 and 515 μm in diameter. To our knowledge, this represents the first report of normal (>50%) pregnancy rates achieved after transfer of cryopreserved expanded equine blastocysts. Our findings suggest that biopsy before cryopreservation, which punctures the embryonic capsule and causes blastocoele collapse, combined with vitrification in a small-diameter pipette, may provide an efficient procedure for cryopreservation of large equine blastocysts. Further studies are needed to explore the different factors associated with success of this procedure. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.