Feral horse population growth rates as high as 25% are of concern to those responsible for managing range lands as well as conservation groups. Current methods to control these populations include adoption and long-term holding, which are both costly and controversial. Porcine zona pellucida (pZP) immunocontraception may have the greatest potential to control fertility because it has proven to be effective in other studies and vaccines are easy and safe to administer. One pZP vaccine formulation, SpayVac 1 (ImmunoVaccine Technologies, Inc., Halifax, NS, Canada), has demonstrated single-dose, multi-year contraceptive efficacy in other wildlife species, which would make it both practical and economical for field application. Over a 7-month period during the breeding season, we assessed the effect on ovarian activity of 2 formulations of SpayVac, 1 non-aqueous with modified Freund's adjuvant (MFA) and the other, an aqueous emulsion with MFA, compared to controls (n ¼ 7 per group). Comparative reproductive parameters included serum concentrations of progesterone (P 4 ) determined by enzyme-linked immunosorbent assay (ELISA), ovarian activity assessed by transrectal ultrasound and palpation, as well as gross and histological examination of ovaries upon necropsy (n ¼ 9 or 3 mares from each group) or after ovariectomy (n ¼ 12 or 4 mares from each group). We determined serum antibody titers using ELISA. Mean serum concentrations of P 4 were less in the non-aqueous MFA treatment group compared to control mares (P < 0.025). Ovaries collected from control mares weighed more (P ¼ 0.002) and had greater variation (P ¼ 0.003) than those from either vaccinated group. Both treatment groups also had smaller ovaries and fewer follicles compared to controls (P < 0.001). Three to 4 months after vaccination, 93% of SpayVac-injected mares ceased cycling; whereas all control mares continued to cycle throughout the study. Relatively constant antibody titers were reached by week 6 post-vaccination, although we found appreciable variation within treatment groups, especially 4-8 weeks post-vaccination. Based on our study, the SpayVac formulations impair ovarian function but do not affect other major organ systems, and could provide a safe and effective immunocontraceptive option for mares. Additional research to elucidate the vaccine's mechanism of action, actual contraceptive efficacy, and long-term effects are still needed. Ó 2013 The Wildlife Society.

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Changes in sodium/potassium adenosine triphosphatase (Na+/K+ ATPase) and Na+/K+ ATPase mRNA content during preimplantation mouse embryo development were determined. Western blotting, using polyclonal antiserum against guinea pig Na+/K+ ATPase, was used to detect changes in Na+/K+ ATPase alpha- and beta-subunit content during mouse embryo development. Total RNA from mouse embryos was analyzed using Northern and slot blots hybridized with random-primer-labeled cDNA for Na+/K+ ATPase alpha-subunit from sheep kidney. Northern blots exhibited a single mRNA band (3.65 kb) in sheep and mouse kidneys and mouse embryos. Although Na+/K+ ATPase alpha-subunit mRNA content of mouse embryos increased 45-fold between Day 1 and Day 4 of development, Na+/K+ ATPase alpha-subunit content remained constant, and beta-subunit content increased 9-fold. The Na+/K+ ATPase alpha-subunit and alpha-subunit mRNA content did not increase in a similar manner. The results suggest that, in mouse embryos, blastocoel formation is not triggered by an increase in Na+/K+ ATPase alpha-subunit content. Changes in beta-subunit content may be important in regulating Na+/K+ ATPase activity and blastocoel formation.

Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6-dimethylaminopurine (6-DMAP), and okadaic acid (OA) on plasminogen activator (PA) activity in porcine oocyte-cumulus cell complexes (POCC) in vitro were determined. Cumulus cell-enclosed oocytes were collected from 1-4 mm antral follicles and cultured in TCM-199 with 0.3% polyvinylpyrrolidone for 48 hr. PA activities in POCC were quantified using SDS-PAGE, casein-agar zymography, and densitometry. Two plasminogen-dependent lytic zones (93-96 kD and 71-79 kD) were observed in POCC. Addition of amiloride to the zymography, a competitive inhibitor of urokinase-type PA, failed to reduce activities in either zone, suggesting that the 71-79 kD band is a tissue-type PA (tPA) and the 93-96 kD band is possibly a tPA-inhibitor complex. Changes in PA activity due to the various treatments were expressed relative to the PA activity in 40 POCC. Increasing dbcAMP increased PA (P < 0.05) activity in dose-dependent fashion, whereas 6-DMAP and 10 and 100 ng/ml PMA inhibited (P < 0.05) PA activity. PA activity increased (P < 0.05) in POCC treated with up to 25 nM OA; however, activity decreased (P < 0.05) at concentrations > 75 nM. Treatment with 25 nM OA also induced the expression of an amiloride-sensitive PA (49-52 kD). Germinal vesicle breakdown and progression to metaphase II were inhibited (P < 0.05) by 2.5 mM dbcAMP and 2 mM 6-DMAP, whereas 100 ng/ml PMA and 25 nM OA inhibited (P < 0.05) only progression to metaphase II.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of plasminogen activator (PA) production to cell stage, cell number and changes in overall diameter and zona pellucida thickness for bovine embryos developing in vitro was determined. Late morulae to blastocysts (n = 80) were collected nonsurgically from naturally mated, estrous-synchronized, superovulated crossbred beef cows. Embryos were cultured, one embryo per 25-microliters microdrop, for 6 d. At 24-h intervals, embryos were evaluated for stage of development and transferred to fresh microdrops; media were recovered for PA analysis. In addition, embryo diameter and zona pellucida thickness were measured with an ocular micrometer. Plasminogen activator production was determined using a caseinolytic assay with urokinase as the standard. Changes in diameter, zona pellucida thickness and PA production per 24-h interval for each embryo were plotted, and the graphs were cut out and weighed. Sixty-one embryos (76%) completed the hatching process. Total PA production was correlated positively (P less than .005) to embryonic size (r = .40), developmental stage (r = .35) and cell number (r = .35) and negatively, but weakly, correlated to zona pellucida thickness (r = -.13; P = .267). Hatched embryos produced more total PA than embryos that did not hatch (.140 +/- .011 vs .070 +/- .019 g; P less than .01). These results suggest that as embryonic size and cell number increase and development progresses, bovine embryos liberate more PA.