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Papers by Gary Killian

Biology of Reproduction, 1990

This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in cult... more This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.

Biology of Reproduction, 1994

The effect of macromolecules from oviductal conditioned medium (CM) on sperm motility and capacit... more The effect of macromolecules from oviductal conditioned medium (CM) on sperm motility and capacitation was studied. Sperm pooled from three bulls was incubated in either luteal isthmic CM, luteal ampullary CM, estrual isthmic CM, estrual ampullary CM, or control medium (no CM) for 4 h. Sperm capacitation and motility were assessed at 10 min and 4 h. Estrual isthmic CM capacitated significantly more spermatozoa at 4 h than estrual ampullary CM or control medium. CM also affected lateral head movement (ALH) and beat cross-frequency (BCF) of sperm. In a second experiment, the glycosaminoglycan (GAG) content of the different types of oviductal CM was quantified. Estrual isthmic CM contained more GAG than estrual ampullary CM. Among luteal samples, no difference in GAG concentration between the isthmic and ampullary CM was found. Heat treatment (100 0 C) of oviductal CM before coincubation with sperm significantly reduced, but did not eliminate, the capacitating ability. Because heat treatment denatures proteins and decreases the capacitating ability of certain GAG, we concluded that the capacitating effect of estrual isthmic CM may be associated with proteins, GAG, and proteoglycans in the CM. Isthmic secretions may play a major role during in vivo sperm capacitation, given that bovine spermatozoa may reside in the oviduct isthmus for up to 18 h before fertilization.

Animal Reproduction Science, 2008

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm w... more The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (␣ L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 • C, 5% CO 2 in air) for 1 h in the following treatments either 500 L of fertilization medium (FM) or FM with ␣ L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h either FM or FM with ␣ L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (␣hist); (3) ␣ L-PGDS at fertilization time (with fertilization medium); (4) ␣ L-PGDS-treated oocytes; or (5) ␣ L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 × 10 4 washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with ␣ L-PGDS: (1) 26.4 ± 3.0; (2) 25.6 ± 3.0; (3) 59.7 ± 3.0; (4) 56.4 ± 3.0; and (5) 57.1 ± 3.0. Addition of ␣ L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 ± 2.0%; (2) 87.5 ± 2.0%; (3) 19.4 ± 2.0%; (4) 27.2 ± 3.1%; and (5) 14.1 ± 3.4%. The ␣ L-PGDS reacts with both oocytes and spermatozoa, resulting in

Animal Reproduction Science, 2005

Animal Reproduction Science, 2000

We conducted a 2-year feasibility study with native porcine zona pellucida (PZP) vaccine and thre... more We conducted a 2-year feasibility study with native porcine zona pellucida (PZP) vaccine and three recombinant rabbit zona pellucida vaccines (RC55, RC75a and a combination of RC55, RC75a and RC75b) as an initial phase of developing a recombinant immunocontraceptive vaccine to control reproduction in overpopulated herds of white-tailed deer (Odocoileus virginianus). Forty captive white-tailed does were divided into five groups (one sham and four treated), of eight each and injected with a 500 g prime dose of vaccine. Each prime dose was followed by a 300 g booster dose at 3-7 weeks post prime. The frequency and number of months of observed breeding were higher in PZP immunized does than in sham controls. Although the antibody titers of the three recombinant groups were 1000 or less, as compared with the PZP group with titers often over 128,000, the fawning rates of the two recombinants were significantly lower than that of the control group. The combined antigen group did not have a significantly lower fawning rate.

Reproduction, Fertility and Development, 2008

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (U... more Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatmen...

Overpopulation of feral horses in several western states is an unquestioned problem. Current mana... more Overpopulation of feral horses in several western states is an unquestioned problem. Current management strategies of removal and adoption are expensive, logistically challenging, and ineffective as a means of population control. We are testing three long-acting contraceptive approaches on feral Nevada mares. Modified reversible immunocontraceptive vaccines for gonadotropin releasing hormone (GnRH) and porcine zona pellucida (F'ZP) (SpayVac), and intrauterine contraceptive devices (IUDs), are being evaluated to determine: I) their safety and efficacy for preventing pregnancy for multiple years, 2) whether the effects are reversible, and 3) whether there are notable contraindications. Preliminruy data after 1 year suggest that IUD- and PZP- treated mares continue to exhibit breeding and estrus, while GnRH-treated mares are less likely to cycle. All mares in the GnRH and PZP treatments were infertile for the breeding season. Eighty percent of the IUD-treated mares were infertile; ...

Reliable activity measures in free-ranging white-tailed deer could be useful for the assessment o... more Reliable activity measures in free-ranging white-tailed deer could be useful for the assessment of estrous cycling and general activity changes that can occur after immunocontraceptive vaccine treatments. Electronic data-logging (CountcardTM) devices attached to 9 white-tailed doe deer on neck collars were used to monitor their movement activities during 2 rutting seasons in 2.5 acre fenced enclosures. Direct daily behavioral observations on buck responses toward individual does were used to detect estrus and to validate changes in 24-hour activity counts as another indication of estrous cycling. When individual activity counts for the estrus days on the does were compared to diestrus counts as estimated by mean values over days 10 through 15 post-estrus, a significant high product moment correlation of 0.946 was obtained indicating similar and proportional changes in activity under the 2 behaviorally-detected conditions. As predicted, mean activity counts for the estrus days were s...

This paper outlines a 25 yr research strategy to better understand how proteins in male and femal... more This paper outlines a 25 yr research strategy to better understand how proteins in male and female reproductive fluids influence fertility. Extensive fertility data available from artificial insemination cooperatives were used to identify individual dairy bulls with phenotypes of above and below average fertility. We determined whether the protein composition of reproductive fluids from these bulls differed with fertility. Surgical alteration of bulls to equip them with indwelling vas deferens cannulae enabled recovery of accessory sex gland fluid and cauda epididymal fluid and sperm for in vitro studies. Extensive analyses of reproductive fluids from these unique individuals enabled identification of several proteins that were associated with fertility. Methods to cannulate the oviducts of cows were also used to evaluate the composition and function of oviductal fluid. In vitro testing of proteins purified from male and female reproductive fluids demonstrated that they facilitated ...

Proceedings of the Vertebrate Pest Conference

The physiological mechanisms controlling ovarian follicular growth and ovulation involve a comple... more The physiological mechanisms controlling ovarian follicular growth and ovulation involve a complex exchange of systemic signals and a localized exchange of molecules between the oocyte and surrounding somatic cells. It has been demonstrated that the oocyte itself plays an essential role in regulating these processes by secreting two key regulatory proteins: bone morphogenetic protein-15 (BMP15) and growth and differentiation factor-9 (GDF9). Natural mutations in their expression and vaccination against these growth factors have been shown to cause sterility in sheep. The aim of this 3-year study was to determine the effect of vaccination against GDF9 and BMP15 on fertility in female white-tailed deer. Does were randomly assigned to two groups (n = 10/group). Each doe received a primary vaccination followed by a booster 47 days later. After the first year, a subset of animals (n = 4-5/group) received an additional booster vaccination. Blood samples were collected at regular intervals to determine antibody titers and progesterone concentrations. Early pregnancy was assessed by ultrasound, and fawning was subsequently monitored. For the BMP15 group, over the 3 years 80%, 100%, and 75% of does fawned with fawning rates of 2.4, 3.5, and 3.3 fawns/doe, respectively. For the GDF9 group, 80%, 25%, and 25% of does fawned with fawning rates of 2.6, 1, and 2 fawns/doe, respectively. The fawning rate of untreated animals in the herd was 1.8 fawns/doe. Although all animals in the BMP15 group had high antibody titers, they were not made infertile, and they became more fecund. Most animals in the GDF9 group had high antibody titers, but fecundity was not affected the first year; however, they were made infertile in Years 2 and 3. Results from this study demonstrate that vaccination against GDF9 has potential to control fertility in deer. Further research will be required to determine the appropriate timing for administering the vaccine and the longevity of effect.

... Killian GJ, 1997: Influence of bovine ampullary and isthmic oviductal fluid on sperm-egg bind... more ... Killian GJ, 1997: Influence of bovine ampullary and isthmic oviductal fluid on sperm-egg binding and fertilization in vitro. J Reprod Fertil 109, 95–101. ... Yang C, Yanagimachi R, 1989: Differences between mature ovarian and oviductal oocytes: a study using the golden hamster. ...

Theriogenology, 2007

Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, ... more Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, has been identified in cow oviduct epithelium and fluid. To investigate the potential role OPN in fertilization, we evaluated the ability of RGD peptide (arginine-glycine-aspartic), RGE peptide (arginine-glycine-glutamic acid), integrins alphaV and alpha5 antibodies and OPN antibody to influence bovine in vitro sperm-egg binding and fertilization. Treatment of sperm or oocytes with the RGD peptide prior fertilization significantly decreased in vitro sperm-egg binding and fertilization compared to the non-treated controls or those treated with RGE peptide. Binding and fertilization were also significantly decreased when in vitro matured bovine oocytes or sperm were pre-incubated with integrins alphaV and alpha5 antibodies at concentration ranging from 5 to 20 microg/mL. Addition of a rabbit polyclonal IgG antibody against purified bovine milk OPN with sperm or/and oocytes decreased (P<...

Reproduction in Domestic Animals, 2008

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous spe... more Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Proceedings of the Vertebrate Pest Conference

GonaCon ™ Immunocontraceptive Vaccine targets the reproductive hormone gonadotropin releasing hor... more GonaCon ™ Immunocontraceptive Vaccine targets the reproductive hormone gonadotropin releasing hormone (GnRH) secreted by the hypothalamus of the brain. Antibodies produced in response to the vaccine inactivate endogenous GnRH, which in turn eliminates stimulation of the pituitary gland and gonads in males or females. The resulting "immunocastration" renders animals unable to produce reproductive steroids or gametes. In previous studies, we demonstrated that GonaCon ™ was effective in impairing fertility for up to 3 years in female deer without apparent side effects. However, detailed post mortem evaluations were not done. To better understand the entire physiological response of deer to GonCon ™ and to establish if contraindications were associated with its use, we undertook the present study. To evaluate toxicity and safety, 7 does were given the standard single injection GnRH-KLH vaccine dose (1000 µg) delivered IM in 1 ml using AdjuVac ™ adjuvant and compared to 6 does given a single control saline IM injection and 6 does given 3 injections of GonaCon ™ at 2-week intervals per dose. The study was conducted for 20 weeks. Does were blocked by weight and randomly assigned within blocks to treatment groups. Blood samples were drawn immediately prior to vaccination and at 5, 10, 15, and 20 weeks post immunization. Blood was assayed for LH, testosterone and progesterone, and anti-GnRH titers, as well as hematology and blood chemistry. At each sampling period, the general health of the doe was observed and the injection site was inspected for the formation of abscesses or other tissue reactions. At Week 20, all deer were euthanized and evaluated at necropsy by veterinary pathologists and samples of lymph nodes, reproductive organs, lung, liver, heart, kidney, spleen, and brain were taken for histology. Aside from granulomata formation at the injection site, there were no significant contraindications or toxic effects associated with GonaCon ™ .

Biology of Reproduction, 2007

Biodiversity and Conservation, 2016

We studied rooting damage during five-years of feral swine control at Avon Park Air Force Range, ... more We studied rooting damage during five-years of feral swine control at Avon Park Air Force Range, a significant botanical biodiversity hotspot in peninsular Florida with many globally imperiled plant species and communities. While control reduced swine abundance, remaining animals consistently rooted the 49 studied sites in both middle-dry season (MDS) and late-dry season (LDS) each year. At each study site, we measured rooting with sub-meter accuracy. Neither total nor proportional area rooted differed in either season, across study years, or among plant community types: herbaceous seepage slopes, wet pine savannas, wet grasslands. The proportion of sites with damage during MDS was at least 25 % less than pre-control baseline. During LDS, the proportion of sites with damage increased over years but remained below the initial 2 years' MDS results. Fresh rooting frequency (rooting \1 week-old) across sites dropped precipitously from baseline and remained low for MDS. Fresh rooting frequency among sites during LDS was lower than MDS for all but year two of the study. Canopied habitat \50 m from a study site almost guaranteed rooting at the site each observation period. We propose actions for protecting wetlands, integrating swine control into other compatible land use practices, and improving swine control efficacy. While we measured damage amounts at each site very accurately, our approach of also considering frequency of rooting and frequency of fresh rooting across sites offers low-labor means to broadly assess swine damage and control Communicated by B. D. Hoffmann.

We determined the effect of a GnRH vaccine on reproductive function of sexually mature 5-month fe... more We determined the effect of a GnRH vaccine on reproductive function of sexually mature 5-month female and male domestic swine. The vaccine, GonaCon TM , developed at NWRC contains a GnRH peptide conjugated to KLH, combined with AdjuVac TM adjuvant also developed at NWRC. Four groups of ten females were given single IM immunizations either of 800µg GnRH vaccine, 1600µg GnRH vaccine, a prime and boost of 400µg GnRH vaccine and a sham dose adjuvant alone (control). At 8 months old, females were evaluated daily for estrus and bred by artificial insemination if in standing heat. Females not showing heat after 60 days were considered infertile. Farrowing data were collected for bred females ~115days later. All control gilts showed heat and became pregnant, whereas none given the dual 400µg dose showed heat or were bred. Of the 10 gilts receiving 800µg GnRH vaccine 3 were bred, but only 2 became pregnant. For gilts receiving 1600µg, 1 showed heat and became pregnant. Serum antibody titers were non-detectable in the control group, highest in gilts receiving 2×-400µg, followed by 1600µg and 800µg treatments. Titers were inversely correlated to suppression of estrus and fertility. Groups of five boars received either adjuvant only, 1×-800µg or 1600µg, or 2×-400µg GnRH vaccine IM. Blood and scrotal testicular measurements were taken initially and 4 and 16week post-treatment. Testicular size and serum testosterone decreased in all groups except controls and was inversely related to serum antibody titers. The authors conclude that; injectable GnRH vaccine is effective for contraception of swine.

Immunocontraceptive vaccines based on gonadotropin-releasing hormone (GnRH) have been tested in a... more Immunocontraceptive vaccines based on gonadotropin-releasing hormone (GnRH) have been tested in adult white-tailed deer (Odocoileus virginianus), but their effects on fawns are unknown. The purpose of this study was to determine if early immunization against GnRH would induce a long-lasting immune response in fawns, and if it would delay or prevent sexual development. We gave primary and subsequent booster injections of a KLH-GnRH/Freund's vaccine to 6 male and 6 female fawns. This vaccine contained the same active ingredients as GonaCon™ vaccine, but it contained Freund's adjuvant instead of AdjuVac™ adjuvant. Two 450-μg injections were given 1 month apart when fawns were 3 and 4 months of age. Although 1 female fawn produced a multiyear contraceptive response similar to that observed previously in vaccinated adults, the immune response lasted only 1 year in the other 11 fawns. Antibody titers in those 11 fawns declined sharply to near zero during the second and third years after vaccination. Our results indicate that treating 3-to 4-month-old white-tailed deer with the GnRH vaccine did not induce contraception or sterilization, and it did not delay or prevent sexual development. No adverse health effects were apparent in GnRH-treated fawns.

Biology of Reproduction, 1990

This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in cult... more This study evaluated the responsiveness of Sertoli cell glycosylation in vitro to changes in culture age and to the presence of a reconstituted basement membrane (Matrigel) or collagen IV/laminin substrata. Primary Sertoli cell cultures were prepared from 20-day-old rats and incubated with [3H]mannose, a monosaccharide specific for asparagine-linked oligosaccharides. The cells were harvested on Days 4, 6, or 10 of culture life. A supernatant enriched in cell-surface glycopeptides (the trypsinate) and a cell pellet stripped of surface glycoconjugates were evaluated separately. Glycopeptides derived from a Pronase digest of the two samples were fractionated using concanavalin-A lectin affinity chromatography into three major classes: multiantennary complex-type, biantennary complex-type, and high-mannose-type oligosaccharide structures. The proportion of radiolabeled glycopeptides appearing in each of the three classes did not differ between Days 4 and 6 of culture. In contrast, a significant increase in the percentage of radiolabeled glycopeptides containing multiantennary complex-type oligosaccharides was observed in cells harvested from the 10-day-old cultures. In other experiments, Sertoli cells were grown on various substrata: plastic; collagen IV/laminin; or Matrigel, a reconstituted basement membrane (RBM) composed of laminin, collagen IV, proteoglycan sulfate, entactin, and nidogen. Growth on RBM significantly increased multiantennary complex-type oligosaccharide formation compared to plastic, whereas the high-mannose-type glycopeptides increased in cells grown on collagen IV/laminin. These studies suggest that environmental and physiological conditions such as culture age and the presence of extracellular matrix significantly affect glycosylation patterns in Sertoli cell cultures.

Biology of Reproduction, 1994

The effect of macromolecules from oviductal conditioned medium (CM) on sperm motility and capacit... more The effect of macromolecules from oviductal conditioned medium (CM) on sperm motility and capacitation was studied. Sperm pooled from three bulls was incubated in either luteal isthmic CM, luteal ampullary CM, estrual isthmic CM, estrual ampullary CM, or control medium (no CM) for 4 h. Sperm capacitation and motility were assessed at 10 min and 4 h. Estrual isthmic CM capacitated significantly more spermatozoa at 4 h than estrual ampullary CM or control medium. CM also affected lateral head movement (ALH) and beat cross-frequency (BCF) of sperm. In a second experiment, the glycosaminoglycan (GAG) content of the different types of oviductal CM was quantified. Estrual isthmic CM contained more GAG than estrual ampullary CM. Among luteal samples, no difference in GAG concentration between the isthmic and ampullary CM was found. Heat treatment (100 0 C) of oviductal CM before coincubation with sperm significantly reduced, but did not eliminate, the capacitating ability. Because heat treatment denatures proteins and decreases the capacitating ability of certain GAG, we concluded that the capacitating effect of estrual isthmic CM may be associated with proteins, GAG, and proteoglycans in the CM. Isthmic secretions may play a major role during in vivo sperm capacitation, given that bovine spermatozoa may reside in the oviduct isthmus for up to 18 h before fertilization.

Animal Reproduction Science, 2008

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm w... more The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (␣ L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 • C, 5% CO 2 in air) for 1 h in the following treatments either 500 L of fertilization medium (FM) or FM with ␣ L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h either FM or FM with ␣ L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (␣hist); (3) ␣ L-PGDS at fertilization time (with fertilization medium); (4) ␣ L-PGDS-treated oocytes; or (5) ␣ L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 × 10 4 washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with ␣ L-PGDS: (1) 26.4 ± 3.0; (2) 25.6 ± 3.0; (3) 59.7 ± 3.0; (4) 56.4 ± 3.0; and (5) 57.1 ± 3.0. Addition of ␣ L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 ± 2.0%; (2) 87.5 ± 2.0%; (3) 19.4 ± 2.0%; (4) 27.2 ± 3.1%; and (5) 14.1 ± 3.4%. The ␣ L-PGDS reacts with both oocytes and spermatozoa, resulting in

Animal Reproduction Science, 2005

Animal Reproduction Science, 2000

We conducted a 2-year feasibility study with native porcine zona pellucida (PZP) vaccine and thre... more We conducted a 2-year feasibility study with native porcine zona pellucida (PZP) vaccine and three recombinant rabbit zona pellucida vaccines (RC55, RC75a and a combination of RC55, RC75a and RC75b) as an initial phase of developing a recombinant immunocontraceptive vaccine to control reproduction in overpopulated herds of white-tailed deer (Odocoileus virginianus). Forty captive white-tailed does were divided into five groups (one sham and four treated), of eight each and injected with a 500 g prime dose of vaccine. Each prime dose was followed by a 300 g booster dose at 3-7 weeks post prime. The frequency and number of months of observed breeding were higher in PZP immunized does than in sham controls. Although the antibody titers of the three recombinant groups were 1000 or less, as compared with the PZP group with titers often over 128,000, the fawning rates of the two recombinants were significantly lower than that of the control group. The combined antigen group did not have a significantly lower fawning rate.

Reproduction, Fertility and Development, 2008

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (U... more Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatmen...

Overpopulation of feral horses in several western states is an unquestioned problem. Current mana... more Overpopulation of feral horses in several western states is an unquestioned problem. Current management strategies of removal and adoption are expensive, logistically challenging, and ineffective as a means of population control. We are testing three long-acting contraceptive approaches on feral Nevada mares. Modified reversible immunocontraceptive vaccines for gonadotropin releasing hormone (GnRH) and porcine zona pellucida (F'ZP) (SpayVac), and intrauterine contraceptive devices (IUDs), are being evaluated to determine: I) their safety and efficacy for preventing pregnancy for multiple years, 2) whether the effects are reversible, and 3) whether there are notable contraindications. Preliminruy data after 1 year suggest that IUD- and PZP- treated mares continue to exhibit breeding and estrus, while GnRH-treated mares are less likely to cycle. All mares in the GnRH and PZP treatments were infertile for the breeding season. Eighty percent of the IUD-treated mares were infertile; ...

Reliable activity measures in free-ranging white-tailed deer could be useful for the assessment o... more Reliable activity measures in free-ranging white-tailed deer could be useful for the assessment of estrous cycling and general activity changes that can occur after immunocontraceptive vaccine treatments. Electronic data-logging (CountcardTM) devices attached to 9 white-tailed doe deer on neck collars were used to monitor their movement activities during 2 rutting seasons in 2.5 acre fenced enclosures. Direct daily behavioral observations on buck responses toward individual does were used to detect estrus and to validate changes in 24-hour activity counts as another indication of estrous cycling. When individual activity counts for the estrus days on the does were compared to diestrus counts as estimated by mean values over days 10 through 15 post-estrus, a significant high product moment correlation of 0.946 was obtained indicating similar and proportional changes in activity under the 2 behaviorally-detected conditions. As predicted, mean activity counts for the estrus days were s...

This paper outlines a 25 yr research strategy to better understand how proteins in male and femal... more This paper outlines a 25 yr research strategy to better understand how proteins in male and female reproductive fluids influence fertility. Extensive fertility data available from artificial insemination cooperatives were used to identify individual dairy bulls with phenotypes of above and below average fertility. We determined whether the protein composition of reproductive fluids from these bulls differed with fertility. Surgical alteration of bulls to equip them with indwelling vas deferens cannulae enabled recovery of accessory sex gland fluid and cauda epididymal fluid and sperm for in vitro studies. Extensive analyses of reproductive fluids from these unique individuals enabled identification of several proteins that were associated with fertility. Methods to cannulate the oviducts of cows were also used to evaluate the composition and function of oviductal fluid. In vitro testing of proteins purified from male and female reproductive fluids demonstrated that they facilitated ...

Proceedings of the Vertebrate Pest Conference

The physiological mechanisms controlling ovarian follicular growth and ovulation involve a comple... more The physiological mechanisms controlling ovarian follicular growth and ovulation involve a complex exchange of systemic signals and a localized exchange of molecules between the oocyte and surrounding somatic cells. It has been demonstrated that the oocyte itself plays an essential role in regulating these processes by secreting two key regulatory proteins: bone morphogenetic protein-15 (BMP15) and growth and differentiation factor-9 (GDF9). Natural mutations in their expression and vaccination against these growth factors have been shown to cause sterility in sheep. The aim of this 3-year study was to determine the effect of vaccination against GDF9 and BMP15 on fertility in female white-tailed deer. Does were randomly assigned to two groups (n = 10/group). Each doe received a primary vaccination followed by a booster 47 days later. After the first year, a subset of animals (n = 4-5/group) received an additional booster vaccination. Blood samples were collected at regular intervals to determine antibody titers and progesterone concentrations. Early pregnancy was assessed by ultrasound, and fawning was subsequently monitored. For the BMP15 group, over the 3 years 80%, 100%, and 75% of does fawned with fawning rates of 2.4, 3.5, and 3.3 fawns/doe, respectively. For the GDF9 group, 80%, 25%, and 25% of does fawned with fawning rates of 2.6, 1, and 2 fawns/doe, respectively. The fawning rate of untreated animals in the herd was 1.8 fawns/doe. Although all animals in the BMP15 group had high antibody titers, they were not made infertile, and they became more fecund. Most animals in the GDF9 group had high antibody titers, but fecundity was not affected the first year; however, they were made infertile in Years 2 and 3. Results from this study demonstrate that vaccination against GDF9 has potential to control fertility in deer. Further research will be required to determine the appropriate timing for administering the vaccine and the longevity of effect.

... Killian GJ, 1997: Influence of bovine ampullary and isthmic oviductal fluid on sperm-egg bind... more ... Killian GJ, 1997: Influence of bovine ampullary and isthmic oviductal fluid on sperm-egg binding and fertilization in vitro. J Reprod Fertil 109, 95–101. ... Yang C, Yanagimachi R, 1989: Differences between mature ovarian and oviductal oocytes: a study using the golden hamster. ...

Theriogenology, 2007

Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, ... more Osteopontin (OPN), a phosphoprotein containing an arginine-glycine-aspartic acid (RGD) sequence, has been identified in cow oviduct epithelium and fluid. To investigate the potential role OPN in fertilization, we evaluated the ability of RGD peptide (arginine-glycine-aspartic), RGE peptide (arginine-glycine-glutamic acid), integrins alphaV and alpha5 antibodies and OPN antibody to influence bovine in vitro sperm-egg binding and fertilization. Treatment of sperm or oocytes with the RGD peptide prior fertilization significantly decreased in vitro sperm-egg binding and fertilization compared to the non-treated controls or those treated with RGE peptide. Binding and fertilization were also significantly decreased when in vitro matured bovine oocytes or sperm were pre-incubated with integrins alphaV and alpha5 antibodies at concentration ranging from 5 to 20 microg/mL. Addition of a rabbit polyclonal IgG antibody against purified bovine milk OPN with sperm or/and oocytes decreased (P<...

Reproduction in Domestic Animals, 2008

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous spe... more Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Proceedings of the Vertebrate Pest Conference

GonaCon ™ Immunocontraceptive Vaccine targets the reproductive hormone gonadotropin releasing hor... more GonaCon ™ Immunocontraceptive Vaccine targets the reproductive hormone gonadotropin releasing hormone (GnRH) secreted by the hypothalamus of the brain. Antibodies produced in response to the vaccine inactivate endogenous GnRH, which in turn eliminates stimulation of the pituitary gland and gonads in males or females. The resulting "immunocastration" renders animals unable to produce reproductive steroids or gametes. In previous studies, we demonstrated that GonaCon ™ was effective in impairing fertility for up to 3 years in female deer without apparent side effects. However, detailed post mortem evaluations were not done. To better understand the entire physiological response of deer to GonCon ™ and to establish if contraindications were associated with its use, we undertook the present study. To evaluate toxicity and safety, 7 does were given the standard single injection GnRH-KLH vaccine dose (1000 µg) delivered IM in 1 ml using AdjuVac ™ adjuvant and compared to 6 does given a single control saline IM injection and 6 does given 3 injections of GonaCon ™ at 2-week intervals per dose. The study was conducted for 20 weeks. Does were blocked by weight and randomly assigned within blocks to treatment groups. Blood samples were drawn immediately prior to vaccination and at 5, 10, 15, and 20 weeks post immunization. Blood was assayed for LH, testosterone and progesterone, and anti-GnRH titers, as well as hematology and blood chemistry. At each sampling period, the general health of the doe was observed and the injection site was inspected for the formation of abscesses or other tissue reactions. At Week 20, all deer were euthanized and evaluated at necropsy by veterinary pathologists and samples of lymph nodes, reproductive organs, lung, liver, heart, kidney, spleen, and brain were taken for histology. Aside from granulomata formation at the injection site, there were no significant contraindications or toxic effects associated with GonaCon ™ .

Biology of Reproduction, 2007

Biodiversity and Conservation, 2016

We studied rooting damage during five-years of feral swine control at Avon Park Air Force Range, ... more We studied rooting damage during five-years of feral swine control at Avon Park Air Force Range, a significant botanical biodiversity hotspot in peninsular Florida with many globally imperiled plant species and communities. While control reduced swine abundance, remaining animals consistently rooted the 49 studied sites in both middle-dry season (MDS) and late-dry season (LDS) each year. At each study site, we measured rooting with sub-meter accuracy. Neither total nor proportional area rooted differed in either season, across study years, or among plant community types: herbaceous seepage slopes, wet pine savannas, wet grasslands. The proportion of sites with damage during MDS was at least 25 % less than pre-control baseline. During LDS, the proportion of sites with damage increased over years but remained below the initial 2 years' MDS results. Fresh rooting frequency (rooting \1 week-old) across sites dropped precipitously from baseline and remained low for MDS. Fresh rooting frequency among sites during LDS was lower than MDS for all but year two of the study. Canopied habitat \50 m from a study site almost guaranteed rooting at the site each observation period. We propose actions for protecting wetlands, integrating swine control into other compatible land use practices, and improving swine control efficacy. While we measured damage amounts at each site very accurately, our approach of also considering frequency of rooting and frequency of fresh rooting across sites offers low-labor means to broadly assess swine damage and control Communicated by B. D. Hoffmann.

We determined the effect of a GnRH vaccine on reproductive function of sexually mature 5-month fe... more We determined the effect of a GnRH vaccine on reproductive function of sexually mature 5-month female and male domestic swine. The vaccine, GonaCon TM , developed at NWRC contains a GnRH peptide conjugated to KLH, combined with AdjuVac TM adjuvant also developed at NWRC. Four groups of ten females were given single IM immunizations either of 800µg GnRH vaccine, 1600µg GnRH vaccine, a prime and boost of 400µg GnRH vaccine and a sham dose adjuvant alone (control). At 8 months old, females were evaluated daily for estrus and bred by artificial insemination if in standing heat. Females not showing heat after 60 days were considered infertile. Farrowing data were collected for bred females ~115days later. All control gilts showed heat and became pregnant, whereas none given the dual 400µg dose showed heat or were bred. Of the 10 gilts receiving 800µg GnRH vaccine 3 were bred, but only 2 became pregnant. For gilts receiving 1600µg, 1 showed heat and became pregnant. Serum antibody titers were non-detectable in the control group, highest in gilts receiving 2×-400µg, followed by 1600µg and 800µg treatments. Titers were inversely correlated to suppression of estrus and fertility. Groups of five boars received either adjuvant only, 1×-800µg or 1600µg, or 2×-400µg GnRH vaccine IM. Blood and scrotal testicular measurements were taken initially and 4 and 16week post-treatment. Testicular size and serum testosterone decreased in all groups except controls and was inversely related to serum antibody titers. The authors conclude that; injectable GnRH vaccine is effective for contraception of swine.

Immunocontraceptive vaccines based on gonadotropin-releasing hormone (GnRH) have been tested in a... more Immunocontraceptive vaccines based on gonadotropin-releasing hormone (GnRH) have been tested in adult white-tailed deer (Odocoileus virginianus), but their effects on fawns are unknown. The purpose of this study was to determine if early immunization against GnRH would induce a long-lasting immune response in fawns, and if it would delay or prevent sexual development. We gave primary and subsequent booster injections of a KLH-GnRH/Freund's vaccine to 6 male and 6 female fawns. This vaccine contained the same active ingredients as GonaCon™ vaccine, but it contained Freund's adjuvant instead of AdjuVac™ adjuvant. Two 450-μg injections were given 1 month apart when fawns were 3 and 4 months of age. Although 1 female fawn produced a multiyear contraceptive response similar to that observed previously in vaccinated adults, the immune response lasted only 1 year in the other 11 fawns. Antibody titers in those 11 fawns declined sharply to near zero during the second and third years after vaccination. Our results indicate that treating 3-to 4-month-old white-tailed deer with the GnRH vaccine did not induce contraception or sterilization, and it did not delay or prevent sexual development. No adverse health effects were apparent in GnRH-treated fawns.