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.

Two experiments were conducted to determine the availability of various vitamin E (E) sources (absorbed to verxite or adsorbed to silica) in broilers. In Experiment 1, at 100 and 300 IU supplemental E, an average of 94 and 44% of E intake from verxite and silica, respectively, was excreted in the feces, but at 30 IU E, 49 and 45% of E intake was excreted in the feces. In Experiment 2 at 30 IU, 52 and 43% of E intake from verxite and silica was excreted (source, P < 0.02). Based on these results, E adsorbed to silica is more available. Two experiments were conducted to determine the relative bioavailability of organic versus inorganic sources of either Mn or Zn. In Experiment 1, Mn as MnSO4 or a Mn amino acid complex (Availa-Mn) were compared. In Experiment 2, ZnSO4 or a Zn amino acid complex (Availa-Zn) were compared. The results indicate that Availa-Mn is a more available source of Mn than MnSO4, but Availa-Zn is not as available as a source of Zn as ZnSO4. An experiment was conducted to determine the effects of organic sources of Zn, Mn, and Cu on White Leghorn pullet performance. Treatment diets consisted of a control diet with 66 ppm Zn as ZnSO4 or a combination of ZnSO4 and Availa-Zn, 66ppm Mn as MnSO4 or a combination of MnSO4 and Availa-Mn, and 10 ppm Cu as CuSO4 or a combination of CuSO4 and a Cu amino acid complex (Availa-Cu). Diets are inorganic sources (IO), organic Zn (OZ), organic Zn and Mn (OZM), or organic Zn, Mn, and Cu (OZMC). Addition of OZM increased (P < 0.08) intestinal tensile strength and increased (P < 0.06) grams of ash per bone compared with OZMC. Pullets fed OZ had a higher (P < 0.08) bone concentration of all minerals than pullets fed OZM. Total tibia Mn was decreased (P < 0.06) by OZMC, but total tibia Cu was increased (P < 0.08) by OZ addition over IO or OZM.

Investigations into the importance of the nuclear integrity of the donor cell prior to nuclear transfer (NT) are limited. In Experiment 1, the proliferative characteristics, chromosomal stability and level of histone phosphorylation in cell lines established by explants and enzymatic dissociation at different population doublings (PDs) were investigated. The cells divided at a constant rate and cell cycle length increased only at the end of the proliferative stage. The level of aneuploidies was high and remained elevated throughout the study independent of the technique used to establish the primary culture. High levels of multinucleated cells and abnormal spindle configurations were observed after prolonged time in culture. An increase in the level of phosphorylated histones occurred after extended time in culture. In Experiment 2, gene expression patterns of chromatin remodeling proteins and levels of DNA methylation and histone acetylation of cells were analyzed. Dnmt-1, MeCP-2 and HDAC-1 expression decreased shortly after establishment of the primary culture. Methylated DNA patterns changed with time in culture, while acetylated histone levels remained constant. Embryo production, developmental potential and gene expression patterns of pre- and post-hatched embryos generated using donor cells with different levels of Dnmt-1 were examined in Experiment 3. A higher proportion of 8-16 cell embryos developed to the blastocyst stage when cells with low levels of Dnmt-1 were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of Dnmt-1 and able to reach the 8-16 cell stage produced a larger number of apparently normal developing embryos, larger conceptuses and higher expression of Dnmt-3a than NT embryos reconstructed using cells with elevated levels of Dnmt-1. However, abnormal gene expression of Dnmts, INFτ and MHC-1 were noted in the majority of cloned embryos indicating inefficient nuclear reprogramming and retarded embryo development. In conclusion, it is likely that the chromosomal abnormalities observed in donor cells at late PDs impair early development of cloned embryos; however, a lower Dnmt-1 content at the time of NT may facilitate the demethylation process during the first divisions resulting in higher development rates in those surviving the 8-16 cell stage.

The objective of this research was to evaluate the effects of various feed additives on growth performance of nursery pigs. An experiment was conducted in which pigs were fed a conventional Phase 1 diet containing: 1) no salmon protein hydrolysate (SPH) or spray-dried porcine plasma (SDPP); 2-3) 1.5% SPH or SDPP; 4-5) 3.0% SPH or SDPP; or 6) 1.5% SPH and 1.5% SDPP. The results of this experiment showed growth performance was unaffected by source or level of protein during any growth phase. Overall gain:feed was greater (P = 0.08) for pigs fed the 1.5% level of protein than for those fed 3.0%, but there was no difference between protein sources. A second experiment was conducted in which pigs were fed a conventional Phase 1 diet (no mammalian protein) containing: 1) no SPH or SDPP; 2-3) 1.5% SPH or SDPP; or 4) 1.5% SPH and 1.5% SDPP. All pigs were fed common Phase 2 and 3 diets. The results of this experiment showed during Phase 1, gain:feed was greater (P < 0.05) for pigs fed SDPP than for those fed SPH. During Phase 2, ADFI was greater (P < 0.05) for pigs fed SPH than for those fed the control. During Phase 3, gain was greater (P = 0.08) for pigs fed SDPP than for those fed SPH. Overall growth performance was unaffected by protein source. An experiment was conducted in which pigs were fed Phase 1, 2, and 3 diets containing: 1) control; 2-5) 10.0% Nutri-Sure (NS), ground oat groats (GOG), ground steam rolled oats (SRO), or feeding oat meal (FOM). The results of this experiment showed during Phase 1, growth performance was unaffected by diet. During Phase 2, feed intake was greater (P < 0.09) for pigs fed GOG or SRO than for those fed NS. During Phase 3, feed intake was greater (P < 0.05) for pigs fed FOM than for those fed the control. Overall gain:feed was greater (P < 0.03) for pigs fed NS or SRO than for those fed the control. The results of these experiments indicate that SPH, SDPP, NS, GOG, SRO, and FOM are adequate additions to nursery diets.

Mares of poor body condition have low plasma leptin concentrations, while obese mares have the highest concentrations. Leptin is a primary signal of body condition to the brain in other species; therefore, low leptin concentrations in thin mares could contribute to their extended anovulatory period in winter compared to obese mares. The current experiment was designed to determine whether recombinant equine leptin, administered to seasonally anovulatory mares, would induce ovarian activity and ovulation in the winter. Leptin effects on metabolism were also studied. Beginning January 7, leptin-treated mares (n = 9) received daily i.m. injections of 10 mg recombinant equine leptin in saline and control mares (n = 10) received equivalent injections of gelatin. Reproductive effects were assessed by daily blood samples and regular ultrasound examination of the ovaries. Weights were also collected routinely. In addition, mares were confined to individual pens and hay consumption was measured as a means of evaluating appetite. A 24-h period of 30-min blood sampling was used to characterize hormone patterns. Over the course of the experiment, there was no difference between groups in follicular activity, date of first ovulation, or prolactin in either daily or frequently collected blood samples. Leptin-treated mares lost more (P < 0.0001) weight than control mares in the first 31 d; however, there was no difference in appetite as indicated by 24-h hay consumption. Post-experimental analysis revealed that leptin-treated mares developed antibodies (P < 0.001) against the injected leptin beginning around d 16. Subsequent leptin treatment did not affect growth hormone secretion during the frequent blood sampling window. Leptin treatment did not affect daily triiodothyronine concentrations; however, treated mares had lower (P < 0.016) daily thyroxine concentrations than control mares. Treated mares also had a tendency (P = 0.11) to have lower insulin concentrations during the frequent sampling period. In conclusion, daily treatment with recombinant equine leptin had an immediate effect on body weight without any effect on hay consumption. Although leptin-treated mares had lower thyroxine concentrations and a tendency for lower insulin, no effect was observed on reproductive endpoints in the time period studied.

The purpose of this research was to determine if increasing levels of RBC would affect growth performance and carcass characteristics of finishing pigs and growth performance of broilers. Three experiments were conducted to determine the effect of incremental levels of red blood cells (RBC; 0 to 4% and 0 to 2%), and thus increasing levels of dietary Leu on growth performance and linear carcass measurements of finishing pigs. Our results suggest that feeding 3 or 4% RBC causes a decrease in growth performance. However, feeding 1 or 2% RBC in the diets of finishing pigs had no detrimental effects on growth performance. Three experiments were conducted to determine the effect of incremental levels of RBC (0, 0.5, 1, 2, 3, 4, 5, 6, and 7%) on growth performance of broilers fed diets with supplemental L-Arg and L-Ile (adequate) and diets with no supplemental L-Arg and L-Ile (deficient). The results of this research indicate that up to 6% RBC can be added to a broiler diet without affecting growth performance as long as the diet is supplemented with L-Arg and L-Ile. Furthermore, up to 3% RBC can be added to broiler diets without supplemental Arg and Ile with no detrimental effects on growth performance. Broilers respond quite differently in growth performance to increasing levels of RBC compared with finishing pigs.