Two experiments studied the effects of pretreatment with estradiol benzoate before treatment with a dopamine antagonist on prolactin secretion and reproductive traits in mares during 1) the seasonal anovulatory period and 2) the normal breeding season. Experiment 1 was performed in winter with 17 mares selected for low follicular activity. Nine mares received estradiol benzoate injections every other day for a total of 10 injections; 8 mares received similar injections of vehicle. Ten days after onset of injections, all mares were placed on a daily injections of sulpiride (250 mg) for 45 d or until ovulation. Plasma prolactin concentrations were higher (P < 0.001) in mares receiving estradiol than in control mares for all assessments from d 12 through 36. Mean day of ovulation was 73.6 d for control mares and 29.0 for estradiol-treated mares (P = 0.016). It was concluded that estradiol treatment greatly enhanced prolactin secretion in response to sulpiride in seasonally anovulatory mares and hastened the date of first ovulation by an average of 45 d. Experiment 2 was designed to assess the efficacy of a single injection preparation of another dopamine antagonist, domperidone, for increasing prolactin secretion in cyclic mares in the summer. The experimental design and procedures used in Experiment 1 were repeated, except that a single shot of 3 g of domperidone was administered on d 11 rather than 45 d of sulpiride injections. Day 0 was the first day of estrus for each mare. Prolactin concentrations were higher (P < 0.05) in mares receiving estradiol than in control mares from d 12 through 25 and after a thyrotropin releasing hormone injection on d 21. It was concluded that estradiol enhanced the prolactin response to a single injection of 3 g of domperidone in cyclic mares in the summer in a manner similar to the estradiol enhancement of prolactin secretion in response to daily sulpiride injections in anovulatory mares in winter. Thus, the single injection of domperidone could possibly replace the daily sulpiride injections used in Experiment 1 to induce ovulation in seasonally anovulatory mares; this needs to be tested in future experiments.

Biological resource banking is becoming important for endangered species conservation. A series of experiments were conducted to address issues concerning collection and utilization of biomaterials from Gulf Coast Native (GCN) sheep (model species) (Ovis aries) and Eland antelope (Taurotragus oryx). In the first experiment, two ejaculates were collected 10 minutes apart from each of five rams three times a week for three weeks to maximize output and minimize handling time. Semen volume, concentration and total number of spermatozoa were significantly greater in first ejaculates, whereas pre-cooled, cooled and post-thaw motility, as well as sperm survival, were greater in second ejaculates. The second experiment was designed to develop a practical method for freezing skin biopsies for tissue culture. Cell survival was enhanced by an equilibration time of at least 15 minutes in biological media (containing blood or serum) as compared to non-biological medium (containing PVP). Then, the possibility of using semen and cooled milk as sources of somatic cells (SC) for in vitro culture was evaluated. Fresh, cooled and cryopreserved semen from rams and fresh eland semen was cultured and although SC plated, proliferation was low. From milk, cell attachment was observed in 92% of the samples, whereas only 38% proliferated in culture. Therefore, the ensuing experiment was focused on increasing in vitro proliferation of semen-derived SC by developing (1) a Percoll gradient technique to separate SC from spermatozoa before culture and (2) a method for co-culture of isolated SC with inactivated mouse fibroblasts. Proliferation was significantly increased by co-culture, but contact between the semen-derived SC and feeder cells was not necessary. Finally, intergeneric nuclear transfer (igNT) of semen-derived eland epithelial cells into bovine cytoplasts showed that these cells could direct early embryonic development up to the 8-cell stage. Determination of BrdU-incorporation and evaluation of nuclear status revealed that initial remodeling of the eland nucleus was similar to that of bovine NT embryos within the first 16 hours post-activation; however, after 84 hours, only 13% of interspecies embryos had cycling nuclei in their blastomeres. Future improvements in the technology should eventually allow cloned offspring to be produced from semen-derived somatic cells.

Haemonchus contortus is one of the most threatening parasites to small ruminant production. Control has traditionally relied on the use of anthelmintics. Control has waned because the worm population has developed resistance to most of the currently available anthelmintics and alternative control measures are needed. Current consumer pressure is to reduce the use of chemicals in agricultural products, thus pushing control methods to more natural and acceptable approaches. One such approach is the feeding of condensed tannin containing forages either as fresh forage or dried products (hay, meal, pellets, etc). This study was conducted to determine the effect of a ground meal form of sericea lespedeza (SL), a forage plant high in condensed tannins, on H. contortus infection in sheep. Twenty-eight mixed sex lambs with essentially zero fecal egg count (FEC) were randomly allocated to 4 treatment groups (7 animals each). Two groups received a bolus of 5000 H. contortus infective larvae (L3) once and the infection was allowed to mature over five weeks (existing infection). The remaining two groups received trickle infections of 500 H. contortus L3 three times a week for three weeks (establishing infection). SL meal was fed over a five week period to one of the existing and one of the establishing infection groups while the other groups were fed bermudagrass hay. All groups were fed bermudagrass hay for an additional two weeks and then necropsied. FEC was significantly reduced in the existing infection SL fed group over the 5 week feeding period. Similarly, FEC was lower in the establishing infection SL fed group, but the difference was not significant. After SL feeding was terminated, FEC increased in both existing and establishing infection groups which indicated an effect on female worm fecundity. At necropsy, there were fewer worms in both SL fed groups, but the differences were not significant. This trend of fewer worm numbers suggested that there may have been an effect on reducing infection level also. These results indicate that SL meal had more of an effect on reducing FEC of existing female worm infections than establishing infections of H. contortus.

The objective of this research was to evaluate color and tenderness in chilled or frozen pork loin chops after antioxidant dipping and modified atmosphere packaging. Loin chops were dipped in 0.3 M calcium chloride, 2.0 % sodium ascorbate, 0.2 M calcium ascorbate, or 0.3 M calcium ascorbate. Non-dipped chops served as controls. Chops were packaged in high oxygen (80% O2 / 20% CO2) or no oxygen (80% N2 / 20% CO2) and stored chilled (4° C) for 7 days or frozen (-18° C) for 21 days. After storage, chops were displayed under continuous fluorescent lighting for 3 or 6 days. Instrumental color evaluations indicated that Longissimus dorsi (LD) L* values (lightness) were not significantly different between treatment combinations. However, chops dipped in 0.2 M or 0.3 M calcium ascorbate, stored frozen, packaged in high oxygen, and displayed for 3 days had higher final LD a* (redness) and b* (yellowness) values than other treatment combinations. Sodium or calcium ascorbate increased a* and b* values in vertebrae bone. Chops frozen for 21 days, dipped in 0.2 M or 0.3 calcium ascorbate, packaged in high oxygen, and displayed for 3 days had higher vertebrae a* and b* values than other treatment combinations. This combination of factors indicates that high oxygen atmospheres along with ascorbate and freezing will help keep the hemoglobin iron in a reduced state. In addition, chops dipped in 0.2 M calcium ascorbate, packaged in high oxygen, frozen for 21 days, and displayed for 3 days had lower percent drip loss, percent cook loss, and shear force values than other treatment combinations. Based on the results of our experiment, dipping pork loin chops in 0.2 M calcium ascorbate, packaging in high oxygen, freezing for 21 days, and displaying for 3 days will enhance color and tenderness.

The objective of this study was to use fluorescent in situ hybridization (FISH) to determine if a skew in the sex ratio was present at the level of the bovine round spermatid. Paraffin embedded bovine testicular tissue obtained from biopsies were used to perform FISH, and the seminiferous tubules were examined. Our findings show that the skew does exist within the round spermatids. Analysis of variance on X- and Y-chromosome bearing round spermatids as affected by sex, slide, picture set, and tubule was performed. Sex showed that the variation in round spermatid sex ratio differed between slides (P = 0.0001). The interaction of sex by slide were indicated to be non-significant (P = 0.0815). Sex within tubule nested in slide showed a significant deviation in sex ratio (P = 0.0122). Picture set showed no significant difference in counts, confirming that the counts were consistent (P = 0.2096). A closer look at the data also showed a 10 to 30% variation in the number of seminiferous tubules with a significant skew per slide. Most the skew was toward the Y-chromosome, but a significant skew toward the X-chromosome was also seen. This skew toward the X-chromosome showed that the skew could be counteracted or reversed. Meiotic drives have been shown to exist in other species. Meiotic drives are alleles with the ability to increase their probability of being transmitted to the next generation. Although we cannot prove that a meiotic drive exists in bovine, our findings agree with the meiotic drive principle. These drives push the sex ratio of the population in one direction. Modifiers attached to these drives can counteract or reverse the effect of the meiotic drive. Environmental factors such as crowding, brought on by intense group housing could also have an effect on the expression of these drives. Further investigation is necessary to completely understand these skews in sex ratio and what factors may affect it.

Recently, interest in the preservation of epididymal sperm as a potential source of valuable genes for genome resource banks has escalated. The development of a successful protocol to recover and cryopreserve sperm harvested from the epididymides would salvage germplasm from genetically valuable males that are injured and can no longer mate or have unexpectedly died and can be used as a model for the preservation of male gametes from endangered species. In a series of experiments, epididymal sperm was successfully harvested, cryopreserved and used for intracytoplasmic sperm injection. In Experiment I, ethylene glycol was found to cause significantly (P<0.05) less osmotic damage to bovine sperm during a one step addition and/or removal at 4°C as compared with glycerol in all concentrations evaluated. Furthermore, prolonged exposure (5 days at 4°C) of ethylene glycol was found to be less toxic than glycerol to sperm. In Experiment II, it was demonstrated that glycerol was more effective than ethylene glycol in providing protection against freezing injury during the cryopreservation process in the concentrations evaluated. In Experiment III, it was demonstrated that epididymal sperm retrieval using seminal plasma is beneficial to enhance sperm overall and progressive motility characteristics and to protect it from morphological abnormalities derived from the freezing process. In Experiment IV, a one step dilution process for removal of glycerol from cryopreserved epididymal sperm was found to significantly affect plasma membrane integrity and mitochondrial function of sperm previously exposed to seminal plasma. However, seminal plasma exposure did not have any significant detrimental effect on acrosome integrity. Furthermore, it was demonstrated that the longevity and survivability in vitro during a 4-hour incubation period at 37°C of post-thaw epididymal sperm exposed to seminal plasma prior to cryopreservation was not compromised when compared with the control extended sperm. In Experiment V, we have demonstrated that fertilization, blastocyst and fetal development could be achieved with cryopreserved bovine epididymal sperm by intracytoplasmic sperm injection (ICSI). To our knowledge, this is the first report in the United States and second in the world to use bovine epididymal sperm for ICSI. We achieved far markedly improved blastocyst rates over those results recently reported in the first study originating in Japan.

Brine incorporation into meat products has become a well established practice in the United States. Brines are used to enhance the eating quality of pork by improving its tenderness and juiciness. Carbon dioxide and carbon monoxide have been used in modified atmosphere packaging as antimicrobial agents and color stabilizers, respectively, to increase the shelf life of retail meats. The objective of this experiment was to analyze the effects of incorporating carbon dioxide and carbon monoxide into brine solutions to subsequently inject into raw chilled pork. Pork loins were injected with brine solutions containing 2.27% phosphates, 3.79% salt and dissolved gas mixtures of 20% CO₂:80% N₂, 80% CO₂:20% CO or 100% CO₂. Pork loins injected with brine solution containing no gas were used as a control. Chops were packaged in high O₂ (20% CO₂:80% O₂) modified atmosphere packaging (MAP), vacuum, no O₂ MAP (20% CO₂:80% N₂), and overwrap packaging and stored for 28 days at 4°C. Pork chops were evaluated every seven days after being displayed under fluorescent light (1200 ± 500 lux) for 48 hrs before evaluation. There were no differences (p ≤ 0.05) in color, percent cook loss, percent drip loss, shear force and total aerobic counts between injection treatments. The change in pH for chops injected with 20% CO₂:80% N₂ and 80% CO₂:20% CO in brine due to the dissolution of carbon dioxide in the tissue was not enough to produce a large bacteriostatic effect. Chops injected with brine containing 80% CO₂:20% CO had increased redness when compared with the other samples. Vacuum, high oxygen MAP and no-oxygen MAP increased the shelf life of the pork chops by lowering bacterial growth and maintaining the quality traits for the length of the study as compared with overwrap packages.

Of the 36 species of felines in the world, all except the domestic cat are listed as endangered or threatened. To preserve the genetic diversity of felines and other species, genome resource banks have been established. Due to limited availability of germ cells for research, studies must use models to optimize the techniques before they are applied to endangered species. In this study, preservation of oocytes and spermatozoa was examined using the bovine as a model for felines. In the first series of experiments, bovine and feline oocytes were dehydrated, vitrified, warmed and cultured to assess their ability to undergo embryonic development using a choline-based medium (CJ2) for vitrification and warming solutions preparation as well as the standard sodium based media. In the second series of experiments, feline spermatozoa were dehydrated using air- and freeze-drying as alternative methods to standard cryopreservation. Assessment was done by examining embryonic development after intracytoplasmic sperm injection (ICSI) and DNA integrity of the dehydrated spermatozoa using the comet assay. In the second series of experiments, bovine and feline oocytes behaved osmotically in response to increasingly concentrated solutions. However, vitrified-warmed bovine oocytes had significantly higher cleavage and blastocyst rates compared with their feline counterparts and development using CJ2 medium was similar to the standard media used for cattle but was detrimental to feline oocytes. In the third experiment, cleavage and blastocyst development of feline oocytes injected with cat spermatozoa preserved using air- and freeze-drying was observed. Also, exposure to the dehydration solution and vitrification did not induce DNA damage but the process of freeze-drying did have significantly higher levels compared with controls. Air-dried sperm did not decondense. In conclusion, the use of bovine oocytes as a model for feline oocytes was successful. Both bovine and feline oocytes responded similarly to dehydration and vitrification, except when processed using CJ2 medium. Furthermore, feline spermatozoa can be preserved using dehydration as demonstrated by their ability to produce blastocysts. This study has encouraging results for germ cell preservation. However, the efficiency of these procedures must be improved before they can be used as alternative methods of preservation in endangered species.

The efficacy of a novel plasmid delivery system was assessed for long-term expression of gonadotropin releasing hormone (GnRH) and growth hormone releasing hormone (GHRH) in horses and goats. The efficacy of the technology was demonstrated using 3 novel plasmids: pSEAP [expressing secreted embryonic alkaline phosphatase (SEAP)], pGHRH (expressing GHRH), and pGnRH (expressing GnRH). Geldings were electroporated with a reporter plasmid expressing SEAP in 3 muscle sites. Expression of SEAP, measured from jugular plasma samples, indicated muscle specificity for uptake and expression of the plasmid. Concentrations of SEAP were greatest (P < 0.05) after pectoralis injection, which was chosen as the site for electroporation in subsequent studies. In a second experiment, stallions were electroporated with pGHRH or pSEAP to evaluate the effect of long-term GHRH treatment on the growth hormone (GH) axis and testicular function. Stallions treated with pGHRH had increased (P < 0.05) plasma concentrations of IGF-I, increased (P < 0.05) volume of accessory sex gland fluid, and increased (P < 0.05) number of normal spermatozoa in the ejaculate relative to controls. In the third experiment, stallions were electroporated with pGnRH or pSEAP to test the effects of GnRH on the reproductive axis. Treatment with pGnRH increased (P < 0.05) plasma testosterone concentrations to d 56 and increased (P < 0.01) the LH response to GnRH on d 21, but did not alter (P > 0.1) seminal characteristics evaluated after 36 d of treatment. In a final experiment, goat does were treated with pGnRH or pSEAP to assess the effects of GnRH treatment on the reproductive axis during seasonal anestrus. Plasma concentrations of LH and FSH were not affected (P > 0.1) by treatment through d 56. Plasma progesterone measurements indicated that ovulation did not occur in does treated with pGnRH or pSEAP. Does treated with pSEAP had increased (P < 0.05) plasma SEAP concentrations. In conclusion, electroporatic plasmid delivery of peptide hormones may serve as an effective technique for expression of protein hormones in the horse and goat.

A series of experiments were conducted to evaluate the potential role of leptin in bovine reproduction. In Experiment 1, mean circulating leptin concentrations of postpartum cows were not affected by exogenous dexamethasone treatments. In Experiment 2, mean leptin concentrations were not correlated with female age or body weight but were positively correlated with body condition scores of beef cattle. Leptin concentrations were higher in 1 year old heifers (8.9 ng/ml) compared with 2 year old cows (6.0 ng/ml), but heifer leptin concentrations were not different than 4 to 6 year old cows (8.0 ng/ml) and cows ≥7 years of age (10.5 ng/ml). Mean leptin concentrations were negatively correlated with age in heifers and cows ≤2 years of age and positively correlated with age in cows >3 years of age. In Experiment 3, there were no differences in mean leptin concentrations for 56 days starting 14 days following AI among 2-year old and 3-year old cows pregnant to AI (1.2 ng/ml), the clean-up bulls (1.2 ng/ml) and nonpregnant females (2.2 ng/ml) after a 60-day breeding season. Plasma leptin concentrations were lower for lactating cows (1.0 ng/ml) compared with nonlactating cows (2.1 ng/ml). Female age did not affect circulating leptin concentrations. In Experiment 4, oviduct and uterine epithelial cells from mid-luteal phase females stained positive for the long form of the leptin receptor, and uterine biopsies revealed intense staining for the long form of the leptin receptor on the luminal side of the uterine endometrium. Bovine blastocysts stained positive for the long form of the leptin receptor in the trophoblast cells. In Experiment 5, addition of leptin to culture medium at 0, 100 and 1,000 ng/ml did not affect the percentage embryos developing to the blastocyst stage. Also, leptin did not affect the ratio of blastocysts:8- to 16-cell embryos among the 0 ng/ml treatment group, the 100 ng/ml treatment and the 1,000 ng/ml treatment groups. Results indicate that in the beef cow, the release of leptin and subsequent role(s) of leptin in reproductive processes are likely different than those that have been reported for mice, rats and humans.