As in vitro fertilization (IVF) becomes more acceptable for the treatment of human infertility, it is essential for clinics to have in place an optimal embryo culture system. The objective of this study was to evaluate whether freezing and storing human embryo culture medium will alter embryo post-thaw viability. In consecutive experiments, commercial culture medium was frozen at -20°C and at -80°C and stored at 4°C until its use in IVF. Each IVF-derived replicate of bovine embryos contained a laboratory control group, a fresh commercial culture medium group(s) and a frozen-thawed commercial culture medium group(s). The commercial culture medium was supplemented with 15% of BSA for days 1 through 4 of in vitro culture and on day 4 was supplemented with 9% of glucose. Embryos were cultured to day 8 of the experiment and then embryo development was evaluated and morphology was evaluated using the RED Score system. In Experiment 1, zygotes (n=2,094) were in vitro cultured in medium that had been frozen (-20°C) with storage times ranging from 3 days to 10 weeks. It was found that embryo culture medium frozen at -20°C and stored for up to 10 weeks did not cause a decrease in 8- to 16-cell embryo or blastocyst development for those embryos cultured in medium frozen for 3 days to 6 weeks over the fresh medium (0 weeks). In Experiment 2, zygotes (n=3,727) were in vitro cultured in both fresh and frozen commercial culture medium (-80°C) that was stored for 0, 1 or 2 weeks before use. This study indicated embryo culture medium frozen at -80°C stored for up to 2 weeks did not cause a decrease in 8- to 16-cell embryo or blastocyst development over that of the fresh medium (0 week). In summary, it was concluded that there was no difference in the blastocyst development rate or morphology at the end of the culture period in medium frozen at -20°C for up to 10 weeks or in medium frozen at -80°C for up to 2 weeks.

Eight Holstein calves approximately 6 months of age (mean BW 185.15 + 16.16 kg) were used in a replicated 4 x 4 Latin Square experiment to study effects of protein sources on performance of weaned dairy calves. Dietary treatments consisted of 16% CP diets with three sources of ruminally undegradable protein (RUP). Experimental diets were corn-silage based, with soybean meal (SBM) as the source of ruminally degradable protein (control) and 3 sources of RUP including heat treated soybean meal (SBM) (SoyPLUS®), animal protein blend (PRO-LAK), and extruded-expelled SBM, included at 45% of the dietary CP. The animals were fed their respective diets twice daily ad libitum. Animals were housed in individual stalls for 14 days for dietary adjustment and feed intake measurements. Steers were housed in metabolism crates during the last 4 days of each experimental period for sample collection. Total fecal and urine output was collected, weighed, and sampled for laboratory analysis of nitrogen during the 4-d collection period. On day 4 of the collection period, animals were fitted with jugular catheters. Blood samples were collected at 15-minute intervals for 6 hours for analysis of growth hormone. An additional blood sample was collected at time 0 for plasma urea nitrogen (PUN) and at 30-minute intervals for IGF-I and insulin. On day 18 of each experimental period body weight, wither height, hip height, and body length were measured. Treatment did not affect dry matter intake (P > 0.05). There were also no effects (P > 0.05) of protein source on nitrogen metabolism, PUN, or growth parameters. There was no effect (P > 0.05) of protein source on GH levels. There was no effect of RUP sources on plasma IGF-I concentrations on (P > 0.05). Treatment did not affect (P > 0.05) insulin concentrations. However, there was an affect of time on insulin concentrations (P < 0.05). There was no difference (P >0.05) among treatments in the digestibility of DM, OM, NDF, ADF, or CP. These data suggest that feeding diets with sources of RUP does not improve performance in weaned dairy calves.

Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions presented to embryos. Since the culture environment is a common aspect of both in vitro production (IVP) and nuclear transfer (NT), research focusing on the in vitro culture system will have the potential to improve both techniques. This study investigated the effects of different culture systems and protein sources on the developmental competence of IVP embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of oct-4, nanog, connexin 43, and GLUT-1 transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium (KSOMaa) supplemented with amino acids. Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, oct-4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of oct-4 in KSOMaa or KSOMaa with CS blastocysts was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 was not expressed at detectable levels in in vivo embryos analyzed in our studies. Connexin 43 was not detected in IVP blastocysts used in Experiment 1. Connexin 43 was detected in KSOMaa, SOFaa, and SOFaa with CS blastocysts in Experiment 2. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT-1 when compared with other treatments and in vivo embryos. Overall, the transcript levels of the majority of the genes analyzed were significantly altered by an in vitro culture condition. Differences continue to be observed between in vitro cultured and in vivo embryos, and until these differences are minimized, aberrations in in vitro development will continue to arise.

The purpose of this research was to determine the optimal level of crystalline amino acid (AA) supplementation that supports maximum growth performance in broilers, and to determine the Gly requirement in broilers. Treatments were replicated with a minimum of 7 pens with 6 broilers per pen. Experiments (Exp.) were conducted from 0- to 18- days (d) post-hatching in brooder batteries. Three Exp. were conducted to determine the maximum level of L-Lysâ¢HCl that could be supplemented to corn-soybean meal (C-SBM) diets without negatively affecting growth performance of broilers. The results of these Exp. indicate that broilers can achieve maximum growth performance when fed a C-SBM diet supplemented with 0.25% L-Lysâ¢HCl. Two Exp. were conducted to determine the fifth, sixth, and seventh limiting AA in a low crude protein (CP), C-SBM diet in broilers. The results of these Exp. indicate that Arg and Val may be equally limiting followed by Ile in a C-SBM diet supplemented with DL-Met, L-Lysâ¢HCl, L-Thr, and Gly. Three Exp. were conducted to determine the Gly requirement of broiler chicks fed a low CP, AA-supplemented diet. The results of this research indicate that 2.078% total Gly + Ser is required for broilers fed a C-SBM diet supplemented with 0.25% L-Lysâ¢HCl.

An asymmetric distribution of the sexes within the left and right uterine horns has been described in multiple polytocous, laboratory species. A series of experiments were conducted to evaluate the sex ratio (% male) of calves gestated in the left and right uterine horns, as well as the sex ratio of embryos originating from the left and right ovaries of cattle. In Experiment 1, the sex ratio of calves and fetuses gestated in the left and right uterine horns was investigated. The sex ratio of calves and fetuses gestated in the right uterine horn was significantly higher compared with the sex ratio of calves and fetuses gestated in the left uterine horn. In addition, the sex ratio of the left and right uterine horns differed significantly from parity. In Experiment 2, embryo transfer data were analyzed in an effort to determine if sex-specific selection pressures were applied to embryos in the uterine horn of transfer. The sex ratio of ET calves born following transfer to the left and right uterine horns was not significantly different. Similarly, the overall sex ratio of calves born in this experiment did not differ significantly from parity. The sex ratio of embryos recovered from the left and right uterine horns of superovulated beef cows was evaluated in Experiment 3. The proportion of male embryos collected from the right uterine horns was significantly greater than from the left uterine horns. The sex ratio of embryos recovered from the uterine horns in this experiment was not different from parity. In an effort to determine the role of interovarian communications in the sex selection process, the sex ratio of embryos recovered from unilaterally ovariectomized superovulated beef heifers was investigated in Experiment 4. The sex ratios of embryos recovered from left- and right-ovary intact heifers were not significantly different. In Experiment 5, the sex ratio of IVP embryos was evaluated. The sex ratio of the IVP embryos was significantly lower than parity and was not different between ovary of origin. In addition, length of time in maturation was determined not to influence the sex ratio.

This research was conducted to determine the effects of pre-incubation warming on the hatchability of post-peak egg production broiler breeder eggs. An experiment with six trials was conducted with 7,920 freshly laid eggs from Ross 308 and 708 broiler breeders from 61-67-wks of age. For each trial, 1,320 eggs were used to determine if pre-incubation warming treatments of 0, 2, 4, and 6, or 0, 3, 6, and 9, or 0, 9, 12 and 15 hrs (at 37.6°C) could improve the hatchability of eggs stored (at 15.5°C) for three days. After a storage period of three days, the eggs were incubated for 21d. Unhatched eggs were broken to determine fertility, and if fertile, stage of embryonic death. Time of hatch was observed for pre-incubation warming treatments 0, 9, 12, and 15 hrs. Of the chicks that hatched, two trials were conducted, each using 192 randomly selected males to determine if the pre-incubation warming treatments affected initial weight, final weight, average daily gain, or feed conversion ratio. Statistical significance was assessed at P < 0.05. Pre-incubation warming of 6 hrs or less did not significantly affect fertile hatchability, total hatchability, embryonic morality or pips. However, pre-incubation warming of 15 hrs negatively affected early-dead mortality (11.6%) when compared to eggs that did not receive any pre-incubation warming (8.7%). Pips were significantly reduced in eggs that were treated for 9, 12 and 15 hrs (1.4, 1.3, and 0.3%, respectively) when compared to eggs that did not receive pre-incubation warming (2.4%). Average hatch time was shortened by pre-incubation warming of 9, 12, and 15 hrs with differences of 5, 7, and 12 hrs, respectively, compared to the eggs that were not pre-incubated. Average final weight, average daily gain, and feed conversion ratio were not significantly different at the end of an 18d trial period. The results of this study provide evidence that pre-incubation warming within a range of 2-15 hrs does not improve hatchability of post-peak broiler breeder eggs when stored for three days. The most significant finding is that eggs can be pre-warmed at incubation temperature for 15 hrs without negatively affecting hatchability.

Plasma leptin concentrations in obese adult horses have been shown to vary widely, and horses tend to fit into two groups: low leptin (<10 ng/mL) and hyperleptinemic (10 to 50 ng/mL). Observations over time revealed that the hyperleptinemic condition was consistent, possibly indicating a relatively permanent underlying cause. Based on these observations, three experimental approaches were used to further study equine obesity-related hyperleptinemia. The first experiment determined the prevalence of hyperleptinemia among postpartum, lactating mares, evaluated its consequence on their re-breeding success, and investigated correlations between leptin levels in lactating and non-lactating mares. Postpartum mares (n = 198) and non-foaling mares (n = 31) were categorized based on their leptin status: normoleptinemic or hyperleptinemic. Leptin in the lactating mares averaged 4.8 ng/mL, and 11 of the 198 (13%) displayed hyperleptinemia. Leptin in the non-lactating mares averaged 7.5 ng/mL, with 9 mares (29%) displaying hyperleptinemia. Of the 198 lactating mares bred, 81% became pregnant; there was no effect of leptin status on re-breeding success. To study one possible cause for hyperleptinemia in well-fed horses, a second experiment explored polymorphism(s) within exon 2 of the equine leptin gene. The DNA from five hyperleptinemic and five normal mares of high body condition was used to analyze exon 2 of the leptin gene for polymorphisms. Based on the 10 mares tested, there was no polymorphism in exon 2 of the equine leptin gene; therefore, polymorphism is not a likely explanation for the high vs. low leptin difference. The third experiment explored the possible effects of hyperleptinemia on the endocrine and immune systems. Endotoxin was given to mares and geldings to investigate the role and/or regulation of leptin in the pro-inflammatory cytokine response. Of the endpoints measured, only platelet count differed between normal and hyperleptinemic horses. Endotoxin infusion caused the expected pro-inflammatory cytokine and endocrine responses, but leptin status was not a significant factor for any endpoint. It is concluded that hyperleptinemia in mares is not associated with polymorphism in exon 2 of the leptin gene, does not affect re-breeding rates of foaling mares, and does not alter the endotoxin-induced responses of the endocrine and immune systems.

The ability to cryopreserve epididymal sperm from mature postmortem bucks has long been of interest to both wildlife conservationists and deer ranchers. Increased understanding of the cryobiology of epididymal sperm from a non domestic species, such as White-tail deer, could aid in development of future protocols to assist in the preservation of endangered species. In Experiment I, results showed that after cooling postmortem bull testes for 22 hours, no significant difference was noted between sperm parameters of epididymal sperm collected at room temperature or at a cool enviorment. In Experiment II, it was shown that White-tail deer sperm could be successfully cryopreserved using a bovine freezing protocol. Also, if immediate processing of epididymal sperm is not an option, testes can be held within the scrotum at 10°C to 15°C for up to 24 hours prior to processing the sperm for freezing. In Experiment III, post-thaw normal sperm morphology results show that glycerol should be considered over DMSO when freezing White-tail deer epididymal sperm. In Experiment IV, it was shown that White-tail deer epididymal sperm can be held in the presence of glycerol for up to 12 hours and still result in post-thaw motility values >30%. Also, when comparing exposure time of White-tail deer epididymal sperm to glycerol there was relatively no change in post-thaw membrane integrity from 0 hours to 24 hours. In the final experiment, the fawning rates of three different estrous synchronization protocols using timed artificial insemination were compared over two consecutive breeding seasons. The most preferred synchronization protocol was a 14 day CIDR with an eCG injection at the time of CIDR removal. A preliminary experiment was then conducted using the 14 day CIDR with eCG to synchronize and artificially inseminate six does with frozen-thawed epididymal sperm, resulting in five healthy fawns from three pregnancies.

The purpose of this research was to determine the influence of dietary Se on glutathione peroxidase (GPx)-1 and 3 activities and relative liver GPx mRNA levels in growing Holstein bull calves. Calves (n = 14) were started 28 d after birth on either a Se adequate (0.15 ppm Se) or deficient (0.01 ppm Se) diet consisting of 3 growth phases and maintained on the diet until 180 d of age. Blood samples were taken from each calf for determination of GPx-1 and GPx-3 activity. Three calves were euthanized at d 21 of age for determination of baseline liver GPx-1 mRNA level. Four calves from each treatment were euthanized at d 180 of age for determination of liver GPx-1 relative mRNA level. Feed intake and average daily gain were not affected by Se level. Mean liver Se concentration was higher (P < 0.05) for baseline calves and those fed the Se adequate diet than for calves fed the Se-deficient diet, but there was no difference between baseline calves and Se adequate calves with respect to liver Se concentration. The GPx-1 activity was greater for Se adequate than Se-deficient calves (P < 0.01) but not until d 84 of age. The GPx-3 activity was considerably more variable than that of GPx-1 with respect to the trend observed for activity by day, and the GPx-3 activity of the Se-deficient group was only less than that of the Se adequate group (P < 0.05) on d 180. N-fold differences were calculated for relative GPx-1 mRNA levels between treatments. There was a 50% decrease in GPx-1 mRNA for Se-deficient calves (P < 0.05) compared with the Se adequate calves. Regression analysis also was performed to determine the relationship between the various response variables. There was only a moderate relationship (r2 = 0.58) between GPx-1 mRNA transcript levels and GPx-1 activity at d 180, despite a correlation coefficient of 0.76. The relationship between GPx-1 mRNA transcript level and GPx-3 activity at d 180 was much stronger (r2 = 0.81), with a correlation coefficient of 0.90, which was unexpected, as GPx-3 is generally considered a short-term indicator of Se status and therefore a much more variable response. Erythrocyte GPx-1 activity was much more sensitive to Se in the diet and thus reflected the diet more closely than did GPx-3. However, GPx-3 activity was more highly correlated to GPx-1 transcript levels. These unexpected results suggest that another trial utilizing larger sample sizes and serial sampling of liver tissue with the sampling of plasma and erythrocytes may provide a clearer picture of the relationship between liver GPx-1 mRNA , tissue Se concentration, and GPx enzyme activities in neonatal and growing Holstein calves.

In recent years, the use of genetic markers has become more and more prevalent in beef breeding programs. This research focused on four previously identified single nucleotide polymorphisms in a leptin gene on chromosome 4 of beef cows. The SNP were E2FB, T945M, UA1, and UA2. Beef cows used in this research were maintained at the Louisiana State University AgCenter Central Research Station. Cows consisted of purebred Bos tauras and Bos indicus cattle as well as crossbreds. The objectives were to estimate genotypic and allelic frequencies for each SNP and to determine the influence of cow breed type, cow age, and SNP genotypes on cow calving rate and date of calving in 2006, cow plasma leptin concentration, body condition score, and pregnancy status in September of 2006, and cow weight change from April to September in 2006. Over all cows, each of the genetic markers showed polymorphism. Allelic frequency for T in these SNP was greater than 0.10. Within cow breed groups the trend for lower or higher frequencies of homozygous genotypes tended to be consistent. Genotypes TT in E2FB, TT in T945M, CC in UA1, and TT in UA2 had lower frequencies. Brahman cows were missing both CT and TT genotypes in UA2 and the UA2‐TT genotype was not present in Braford, Romosinuano F1 and Brahman F1 breed groups. Neither of the genetic marker genotypes influenced variation in plasma leptin level (P > 0.05). Several genetic markers had effects associated with cow traits that were of interest to this research. UA1 genotypes tended (P=0.07) to have an effect on calving rate. UA2 genotypes were associated (P<0.05) with calving date as well as weight loss. Cow breed group influenced (P < 0.05) calving rate, Julian calving date, weight change, and palpation status. Six year and older cows had a larger plasma leptin level and two year old cows had lower body condition scores than other ages of cows. These results indicate that after adjusting for cow breed group and cow age, genetic markers UA1 and UA2 appear to be associated with several reproductive and weight change traits of beef cows.