Influence of an immune-modulatory feed supplement on performance and immune function of beef cows and calves preweaning.

The ability to positively alter immune and stress response with nutritional compounds is of great interest and importance to the beef industry. There is a proprietary product (OmniGen-AF [OG]; Phibro Animal Health, Quincy, IL) reported to have performance-enhancing benefits by altering animal response to stress and immune challenges. The objective of this 2-yr research project was to study the effect of supplementing OG to beef cows and their calves on breeding and growth performance. One hundred and twelve multiparous beef cows and 48 primiparous cows were randomly assigned to treatment in year 1; control (CON, no OG; n = 56 multiparous and 24 primiparous) or treatment (OG fed at 8.8 g/100 kg body weight [BW]; n = 56 multiparous and 24 primiparous). Multiparous cows (mean ± SD = 6.4 ± 0.4 yr; BW = 589 ± 9.2 kg; body condition score [BCS] 6.2 ± 0.07) were used in both years of the experiment and primiparous cows (mean ± SD = 2.1 ± 0.04 yr of age, weighed 400 ± 7.5 kg, and BSC of 5.6 ± 0.06) were only used in the first year of the experiment. CON and OG supplements were offered over two production cycles beginning in December approximately 60 d prior to projected calving through pre-breeding in May of each year. Calves from treatment cows were offered treatments in a creep supplement limited to a daily rate of 1% as-fed of BW prorated for 3-d/wk feeding from mid-July through weaning with OG offered at 8.8 g/100 kg BW. Primiparous cow's BW, BCS, and calf performance were not affected by treatment (P ≥ 0.15) in year 1. BW of multiparous OG cows tended (P = 0.10) to be heavier at weaning in year 1 and was greater (P = 0.05) at the onset of the experiment in year 2. Body condition of OG cows was greater (P ≤ 0.02) at weaning in both years 1 and 2, as well as at the onset of the experiment in year 2. Calves fed OG from the mature cows gained more (P = 0.05) BW during the creep feeding period than CON. Core body temperatures of OG heifers measured during the late summer with intravaginal temperature data loggers tended (P ≤ 0.10) to be less at 1400 and 1700 hours and were less (P = 0.05) at 1800 hours than CON heifers. Feeding OG did not result in changes (P = 0.25) in serum titer response to the BVD virus of calves during year 2. The results of the current experiment indicate feeding OG to beef cows and calves can result in improvement in BCS of cows, enhance weight gain of calves preweaning, and reduce heat loads in heifer calves during the late summer.

A natural bioactive feed additive alters expression of genes involved in inflammation in whole blood of healthy Angus heifers.

A greater demand for food animal production without antibiotics has created the common practice of feeding food animals dietary immunomodulatory feed additives (IFA) throughout their life cycle. However, little is known about the impact of IFA on cytokine and chemokine signaling in non-stressed, non-pathogen-challenged food animals during the early feeding period. We evaluated the expression of 82 genes related to cytokine and chemokine signaling in the whole blood of growing Angus heifers to determine the effect of IFA supplementation on cytokine and chemokine signaling during the first 28 d of feeding. One gene (CCL1) was significantly up-regulated and 14 genes (17%) were significantly down-regulated by IFA feeding during the entire early feeding period including 5 of 21 (24%) evaluated chemokine and IL receptors (CCR1, CCR2, IL1R1, IL10RA, IL10RB). These data when taken together suggest providing an IFA in the diet of growing beef cattle during the early feeding period may suppress the inflammatory response through cytokine-cytokine receptor signaling.

Supplementation of OmniGen-AF improves the metabolic response to a glucose tolerance test in beef heifers.

This study determined whether feeding the immunomodulating supplement, OmniGen-AF, to feedlot heifers would alter metabolic profiles to a glucose tolerance test. Heifer calves (n = 32; 217 ± 2 kg) were allocated into two treatment diets: 1) Control, fed a standard receiving ration, and 2) OmniGen, fed the Control diet supplemented with OmniGen at 4.54 g/45 kg BW/d. Heifers were fed for 42 d. On d 42, Heifers were processed through a working facility for placement of indwelling jugular catheters. After these procedures, heifers were moved into individual stanchions in an enclosed barn and all heifers were fed their treatment diets at 1400 h. All orts were removed at 2000 h to allow for a 12-h fast prior to first blood collection. The following day, heifers were administered 0.5 mL/kg BW of a 50% dextrose solution at 0900 h (0 min). Blood samples were collected for serum isolation at -60, -45, -30, -15, 0, 10, 20, 30, 45, 60, 90, 120, and 150 min relative to bolus dextrose infusion. Serum was stored at -80 oC until analyzed for cortisol, glucose, insulin, non-esterified fatty acid (NEFA) and urea N concentrations. There was a treatment × time interaction for post-challenge cortisol (P = 0.004) such that cortisol was greater in OmniGen heifers than Control heifers from 10- to 45- min post-infusion. Glucose concentrations increased post-infusion (P < 0.01) and were reduced in OmniGen compared to Control heifers at 10-, 45-, and 90-min after challenge (treatment × time P < 0.001). Similarly, there was a treatment × time interaction for post-challenge insulin concentrations (P = 0.04) such that insulin was greater in OmniGen-fed heifers than Control heifers from 10 to 30 min. In addition, there was a treatment × time interaction (P = 0.01) for NEFA concentrations such that concentrations were reduced in OmniGen-supplemented heifers from 10 to 30 min following administration of the dextrose bolus. Serum urea N concentrations were greater in Control heifers at 150 min compared to OmniGen-fed heifers (post-challenge treatment × time interaction: P < 0.001). These data suggest that OmniGen-fed heifers were more responsive to changes in glucose, perhaps affecting the storage and/or redistribution of energy deposits and provide further evidence for altered metabolism in OmniGen-supplemented cattle. The differences observed may explain differences observed in the immune response in OmniGen-supplemented calves.

Effects of feeding an immunomodulatory supplement to heat-stressed or actively cooled cows during late gestation on postnatal immunity, health, and growth of calves.

Heat stress during late gestation negatively affects the physiology, health, and productivity of dairy cows as well as the calves developing in utero. Providing cows with active cooling devices, such as fans and soakers, and supplementing cows with an immunomodulating feed additive, OmniGen-AF (OG; Phibro Animal Health Corporation), improves immune function and milk yield of cows. It is unknown if maternal supplementation of OG combined with active cooling during late gestation might benefit the developing calf as well. Herein we evaluated markers of innate immune function, including immune cell counts, acute phase proteins, and neutrophil function, of calves born to multiparous dams in a 2 × 2 factorial design. Dams were supplemented with OG or a bentonite control (NO) beginning at 60 d before dry off and exposed to heat stress with cooling (CL) or without active cooling (HT) during the dry period (∼46 d). At birth, calves were separated from their dams and fed 6.6 L of their dams' colostrum in 2 meals. Calf body weight and rectal temperature were recorded, and blood samples were collected at birth (before colostrum feeding) and at 10, 28, and 49 d of age. Calves born to either CL dams or OG dams were heavier at birth than calves born to HT or NO dams, respectively. Concentrations of serum amyloid A were higher in the blood of calves born to OG dams relative to NO and for HT calves relative to CL calves. In addition, calves born to cooled OG dams had greater concentrations of plasma haptoglobin than calves born to cooled control dams. Neutrophil function at 10 d of age was enhanced in calves born to cooled OG dams and lymphocyte counts were higher in calves born to OG dams. Together these results suggest that adding OG to maternal feed in combination with active cooling of cows during late gestation is effective in mitigating the negative effects of in utero heat stress on postnatal calf growth and immune competence.

Effect of nutritional immunomodulation and heat stress during the dry period on subsequent performance of cows.

Heat stress in dairy cows during the dry period impairs milk yield in the next lactation. Feeding OmniGen-AF (OG; Phibro Animal Health Corp., Teaneck, NJ) to lactating cows during heat stress may increase dry matter intake (DMI) and lowers respiration rate (RR) and rectal temperature (RT), but the effects in dry cows are not known. We hypothesized that OG supplementation before, during, and after the dry period (approximately 160 d total) would overcome the effects of heat stress and improve cow performance in the next lactation. Cows were randomly assigned to OG or control (placebo) treatments for the last 60 d in milk (DIM), based on mature-equivalent milk yield in the previous lactation. Cows were dried off 45 d before expected calving and randomly assigned to heat stress (HT) or cooling (CL) treatments. Thus, cows received dietary supplementation during late lactation before they were exposed to either CL or HT. After dry-off, treatment groups included heat stress with placebo (HT, only shade, 56 g/d of placebo, n = 17), HT with OG supplementation (HTOG, 56 g/d of OG, n = 19), cooling with placebo (CL, shade, fans, and soakers, 56 g/d of placebo, n = 16), and CL with OG supplementation (CLOG, 56 g/d of OG, n = 11). After parturition, all cows were kept under the same CL system and management, and all cows continued to receive OG or control treatment until 60 DIM. Cooling cows during the dry period reduced afternoon RT (CL vs. HT; 38.9 ± 0.05 vs. 39.3 ± 0.05°C) and RR (CL vs. HT; 45 ± 1.6 vs. 77 ± 1.6 breaths/min). Respiration rate was also decreased by OG supplementation under HT conditions (HTOG vs. HT; 69.7 ± 1.6 vs. 77.2 ± 1.6 breaths/min). An interaction was observed between OG supplementation and HT; HTOG cows tended to have lower morning RT compared with HT cows. During the dry period, OG reduced DMI relative to control cows. Birth weight was greater in calves from CL cows (CL vs. HT; 40.6 ± 1.09 vs. 38.7 ± 1.09 kg). No differences were detected among treatments in hematocrit, total protein, and body condition score. Cows offered CLOG, CL, and HTOG treatments had greater body weight during the dry period (794.9 ± 17.9, 746.8 ± 16.7, and 762.9 ± 14.9 kg, respectively) than HT cows (720 ± 16.2 kg). Gestation length was approximately 4 d longer for CL cows compared with HT cows. Cows offered CLOG, CL, and HTOG treatments produced more milk (41.3 ± 1.6, 40.7 ± 1.6, and 40.5 ± 1.6 kg/d, respectively) than HT treatment (35.9 ± 1.6 kg/d). Body weight after parturition and DMI were evaluated up to 60 DIM and averaged 661.5 ± 15.8 and 19.4 ± 0.7 kg/d, respectively, with no differences observed among treatments. These results confirm that exposure of dry cows to heat stress negatively affects milk yield in the subsequent lactation. Active cooling of dry cows and OG supplementation can reduce the negative effects of heat stress in the dry period on subsequent performance.

Yeast-containing feed additive alters gene expression profiles associated with innate immunity in whole blood of a rodent model.

Feeding a yeast-containing additive (YCA; OmniGen-AF) improves immune responses in ruminant livestock and reduces subsequent production losses. The objective was to identify molecular pathways by which dietary YCA may modify immune responses using a rodent model. Thirty-seven healthy, unchallenged CD rats received a diet containing 0 (control; n = 5, only 28 d), 0.5% (n = 15) or 1% (n = 17) YCA for 7 (n = 4/group), 14 (n = 3 or 4/group), 21 (n = 3 or 4/group) or 28 (n = 5/group) d. At the end of the feeding periods, whole blood was collected and the isolated RNA was analyzed for the expression of 84 genes involved in innate and cell-mediated adaptive immune responses. Three bacterial pattern recognition receptors TLR1 (0.5%: + 2.01; 1%: + 2.38), TLR6 (0.5%: + 2.11; 1%: + 2.34) and NOD2 (0.5%: + 2.32; 1%: + 2.23), two APC surface receptors CD1D1 (0.5%: + 1.75; 1%: + 2.33) and CD80 (0.5%: +2.45; 1%: +3.00), and the cell signaling molecule MAPK8 (0.5%: +1.87; 1%: +2.35) were significantly up-regulated by YCA at both inclusion rates. In conclusion, feeding YCA may potentially increase recognition and responses to bacterial pathogens and T-cell activation and differentiation and thereby maintain health and prevent production losses.

The house mouse (Mus musculus L.) exerts strong differential grain consumption preferences among hard red and white spring wheat (Triticum aestivum L.) varieties in a single-elimination tournament design.

Wheat (Triticum aestivum L.) plays a central role in the health and nutrition of humans. Yet, little is known about possible flavor differences among different varieties. We have developed a model system using the house mouse (Mus musculus L.) to determine feeding preferences as a prelude to extending results to human sensory analysis. Here, we examine the application of a single-elimination tournament design to the analysis of consumption preferences of a set of hard red and hard white spring wheat varieties. A single-elimination tournament design in this case pairs 2 wheat varieties and only 1 of the 2 is advanced to further tests. Preferred varieties were advanced until an overall "winner" was identified; conversely, less desirable varieties were advanced such that an overall "loser" was identified. Hollis and IDO702 were the winner and loser, respectively, for the hard red varieties, and Clear White 515 and WA8123 were the winner and loser, respectively, for the hard white varieties. When using the more powerful protocol of 14 mice and a 4-d trial, differences in mean daily consumption preferences of 2 varieties were separated at P-values as small as 2 × 10(-8) . The single-elimination tournament design is an efficient means of identifying the most and least desirable varieties among a larger set of samples. One application for identifying the 2 extremes in preference within a group of varieties would be to use them as parents of a population to identify quantitative trait loci for preference.

Optimizing experimental design using the house mouse (Mus musculus L.) as a model for determining grain feeding preferences.

There is little research evaluating flavor preferences among wheat varieties. We previously demonstrated that mice exert very strong preferences when given binary mixtures of wheat varieties. We plan to utilize mice to identify wheat genes associated with flavor, and then relate this back to human preferences. Here we explore the effects of experimental design including the number of days (from 1 to 4) and number of mice (from 2 to 15) in order to identify designs that provide significant statistical inferences while minimizing requirements for labor and animals. When mice expressed a significant preference between 2 wheat varieties, increasing the number of days (for a given number of mice) increased the significance level (decreased P-values) for their preference, as expected, but with diminishing benefit as more days were added. However, increasing the number of mice (for a given number of days) provided a more dramatic log-linear decrease in P-values and thus increased statistical power. In conclusion, when evaluating mouse feeding preferences in binary mixtures of grain, an efficient experimental design would emphasize fewer days rather than fewer animals thus shortening the experiment duration and reducing the overall requirement for labor and animals.

Transgenerational effects of di-(2-ethylhexyl) phthalate on testicular germ cell associations and spermatogonial stem cells in mice.

Recent evidence has linked human phthalate exposure to abnormal reproductive and hormonal effects. Phthalates are plasticizers that confer flexibility and transparency to plastics, but they readily contaminate the body and the environment. In this study, timed pregnant CD1 outbred mice were treated with di-(2-ethylhexyl) phthalate (DEHP) from Embryonic Day 7 (E7) to E14. The subsequent generation (F1) offspring were then bred to produce the F2, F3, and F4 offspring, without any further DEHP treatment. This exposure scheme disrupted testicular germ cell association and decreased sperm count and motility in F1 to F4 offspring. By spermatogonial transplantation techniques, the exposure scheme also disrupted spermatogonial stem cell (SSC) function of F3 offspring. The W/W(V) recipient testes transplanted with F3 offspring germ cells from the DEHP-treated group had a dramatically lower percentage of donor germ cell-derived spermatogenic recovery in seminiferous tubules when compared to the recipient testes transplanted with CD1 control germ cells. Further characterization showed that the major block of donor germ cell-derived spermatogenesis was before the appearance of undifferentiated spermatogonia. Interestingly, the testes transplanted with the F3 offspring germ cells from the DEHP-treated group, when regenerated, replicated testis morphology similar to that observed in the testes from the F1 to F3 offspring of the DEHP-treated group, suggesting that the germ cell disorganization phenotype originates from the stem cells of F3 offspring. In conclusion, embryonic exposure to DEHP was found to disrupt testicular germ cell organization and SSC function in a transgenerational manner.

Enhanced genetic integrity in mouse germ cells.

Genetically based diseases constitute a major human health burden, and de novo germline mutations represent a source of heritable genetic alterations that can cause such disorders in offspring. The availability of transgenic rodent systems with recoverable, mutation reporter genes has been used to assess the occurrence of spontaneous point mutations in germline cells. Previous studies using the lacI mutation reporter transgenic mouse system showed that the frequency of spontaneous mutations is significantly lower in advanced male germ cells than in somatic cell types from the same individuals. Here we used this same mutation reporter transgene system to show that female germ cells also display a mutation frequency that is lower than that in corresponding somatic cells and similar to that seen in male germ cells, indicating this is a common feature of germ cells in both sexes. In addition, we showed that statistically significant differences in mutation frequencies are evident between germ cells and somatic cells in both sexes as early as mid-fetal stages in the mouse. Finally, a comparison of the mutation frequency in a general population of early type A spermatogonia with that in a population enriched for Thy-1-positive spermatogonia suggests there is heterogeneity among the early spermatogonial population such that a subset of these cells are predestined to form true spermatogonial stem cells. Taken together, these results support the disposable soma theory, which posits that genetic integrity is normally maintained more stringently in the germ line than in the soma and suggests that this is achieved by minimizing the initial occurrence of mutations in early germline cells and their subsequent gametogenic progeny relative to that in somatic cells.

Novel vein closure procedure using a proprietary cyanoacrylate adhesive: 30-day swine model results.

PURPOSE: To conduct a pilot study to demonstrate a novel method of using a proprietary cyanoacrylate (CA) for closure of superficial veins. MATERIALS AND METHODS: Right and left superficial epigastric veins from two swine models were utilized due to the vein's similarities with the human great saphenous vein. Under ultrasound guidance, access was gained and a 5-F delivery catheter was advanced to the junction of the superficial epigastric and abdominus rectus veins. A dispenser gun was then utilized to inject 0.16 mL of CA while compression was applied cephalad to the end of the catheter. Immediately after delivery, the catheter was pulled back 3 cm and manual compression was employed for 30 seconds. After this first injection, the ultrasound probe was repositioned caudad to the injection and cephalad to the catheter tip and another 0.16 mL injection was delivered with immediate 3 cm pullback of the delivery system. Manual compression was applied at the caudad end of the treated vein for 30 seconds. This process was repeated until the entire target segment was treated. RESULTS: At 30 days postimplantation, the treated veins were occluded with no evidence of recanalization or migration. Histological findings revealed that the lumen was dilated by coalescing, arborizing clear spaces with entrapped lytic erythrocytes, demarcated by a thin band of granular eosinophilic material. Spindle cells with dense eosinophilic matrix replaced the tunica intima and disrupted the tunica media. CONCLUSION: Results of this initial study demonstrated that intravascular injection of CA is feasible for closure of superficial veins in animal models. These findings warrant further animal studies of this proprietary CA to assess efficacy, safety and its effects on perivenous structures.

Endocrine regulation of spermatogonial stem cells in the seminiferous epithelium of adult mice.

A balance between self-renewal and differentiation of spermatogonial stem cells (SSCs) is required to maintain sperm production throughout male life. The seminiferous epithelium is organized into stages of spermatogenesis based on the complement of germ cell types within a tubular section of the testis. The stages exist in close physical proximity and foster diverse phases of germ cell development despite exposure to a similar endocrine milieu that supports coordinated spermatogenesis. The objective of the current study was to identify the population dynamics of SSCs in vivo. We hypothesized that SSC populations and their niches are specifically distributed across the mature seminiferous epithelium in the mouse testis. To test this hypothesis, we conducted stem cell transplantation of germ cells obtained from stage-specific clusters of seminiferous tubules representing areas of high responsiveness to follicle-stimulating hormone (IX-I), androgen (II-IV), and retinoid (V-VIII) signaling. Similarly, we analyzed the expression of genes linked with SSC activity in these groups of stages. No stage-specific differences in the colonization efficiency or the colony number were detected after SSC transplantation, indicating that SSCs are equally distributed across all stages of the seminiferous tubule. In contrast, SSCs obtained from donor stages IX-IV established larger donor-derived colonies due to increased colony expansion. SSCs originating from different stages have varying degrees of stem cell activity in vivo, a notion consistent with Gdnf, Ret, and Bcl6b expression data. These results support the conclusion of a stage-specific, microenvironment-regulating SSC self-renewal and suggest the presence of a transit-amplifying population of undifferentiated spermatogonia in vivo.

VEGFA family isoforms regulate spermatogonial stem cell homeostasis in vivo.

The objective of the present study was to investigate vascular endothelial growth factor A (VEGFA) isoform regulation of cell fate decisions of spermatogonial stem cells (SSC) in vivo. The expression pattern and cell-specific distribution of VEGF isoforms, receptors, and coreceptors during testis development postnatal d 1-180 suggest a nonvascular function for VEGF regulation of early germ cell homeostasis. Populations of undifferentiated spermatogonia present shortly after birth were positive for VEGF receptor activation as demonstrated by immunohistochemical analysis. Thus, we hypothesized that proangiogenic isoforms of VEGF (VEGFA(164)) stimulate SSC self-renewal, whereas antiangiogenic isoforms of VEGF (VEGFA(165)b) induce differentiation of SSC. To test this hypothesis, we used transplantation to assay the stem cell activity of SSC obtained from neonatal mice treated daily from postnatal d 3-5 with 1) vehicle, 2) VEGFA(164), 3) VEGFA(165)b, 4) IgG control, 5) anti-VEGFA(164), and 6) anti-VEGFA(165)b. SSC transplantation analysis demonstrated that VEGFA(164) supports self-renewal, whereas VEGFA(165)b stimulates differentiation of mouse SSC in vivo. Gene expression analysis of SSC-associated factors and morphometric analysis of germ cell populations confirmed the effects of treatment on modulating the biological activity of SSC. These findings indicate a nonvascular role for VEGF in testis development and suggest that a delicate balance between VEGFA(164) and VEGFA(165)b isoforms orchestrates the cell fate decisions of SSC. Future in vivo and in vitro experimentation will focus on elucidating the mechanisms by which VEGFA isoforms regulate SSC homeostasis.

Acute ethanol exposure affects spermatogonial stem cell homeostasis in pre-pubertal mice.

Ethanol is a known modulator of neural stem cell development, but the consequences of ethanol toxicity on the cell fate decisions of spermatogonial stem cells (SSCs) is poorly understood. Using an in vivo treatment and stem cell transplantation approach, we investigated the effects of acute ethanol exposure on formation of the growing adult SSC population in neonatal and pre-pubertal mice. Treatment with a single dose of ethanol disrupted SSC homeostasis in vivo evidenced by a significant reduction (7-fold) of stem cell colonization efficiency in the testes of recipient mice following transplantation. Ethanol treatment also increased the rate of apoptosis in adult differentiating germ cells in situ. Gene expression analysis indicates that ethanol exposure has transient and long-term effects on the expression of GDNF and VEGF family molecules and supports the hypothesis that the niche microenvironment for SSCs is sensitive to ethanol toxicity during pre-pubertaland adult life.

Water buffalo genome science comes of age.

The water buffalo is vital to the lives of small farmers and to the economy of many countries worldwide. Not only are they draught animals, but they are also a source of meat, horns, skin and particularly the rich and precious milk that may be converted to creams, butter, yogurt and many cheeses. Genome analysis of water buffalo has advanced significantly in recent years. This review focuses on currently available genome resources in water buffalo in terms of cytogenetic characterization, whole genome mapping and next generation sequencing. No doubt, these resources indicate that genome science comes of age in the species and will provide knowledge and technologies to help optimize production potential, reproduction efficiency, product quality, nutritional value and resistance to diseases. As water buffalo and domestic cattle, both members of the Bovidae family, are closely related, the vast amount of cattle genetic/genomic resources might serve as shortcuts for the buffalo community to further advance genome science and biotechnologies in the species.

A global view of 54,001 single nucleotide polymorphisms (SNPs) on the Illumina BovineSNP50 BeadChip and their transferability to water buffalo.

The Illumina BovineSNP50 BeadChip features 54,001 informative single nucleotide polymorphisms (SNPs) that uniformly span the entire bovine genome. Among them, 52,255 SNPs have locations assigned in the current genome assembly (Btau_4.0), including 19,294 (37%) intragenic SNPs (i.e., located within genes) and 32,961 (63%) intergenic SNPs (i.e., located between genes). While the SNPs represented on the Illumina Bovine50K BeadChip are evenly distributed along each bovine chromosome, there are over 14,000 genes that have no SNPs placed on the current BeadChip. Kernel density estimation, a non-parametric method, was used in the present study to identify SNP-poor and SNP-rich regions on each bovine chromosome. With bandwidth = 0.05 Mb, we observed that most regions have SNP densities within 2 standard deviations of the chromosome SNP density mean. The SNP density on chromosome X was the most dynamic, with more than 30 SNP-rich regions and at least 20 regions with no SNPs. Genotyping ten water buffalo using the Illumina BovineSNP50 BeadChip revealed that 41,870 of the 54,001 SNPs are fully scored on all ten water buffalo, but 6,771 SNPs are partially scored on one to nine animals. Both fully scored and partially/no scored SNPs are clearly clustered with various sizes on each chromosome. However, among 43,687 bovine SNPs that were successfully genotyped on nine and ten water buffalo, only 1,159 were polymorphic in the species. These results indicate that the SNPs sites, but not the polymorphisms, are conserved between two species. Overall, our present study provides a solid foundation to further characterize the SNP evolutionary process, thus improving understanding of within- and between-species biodiversity, phylogenetics and adaption to environmental changes.

Vascular endothelial growth factor regulates germ cell survival during establishment of spermatogenesis in the bovine testis.

Vascular endothelial growth factor-A (VEGFA) is a hypoxia-inducible peptide essential for angiogenesis and targets nonvascular cells in a variety of tissues and cell types. The objective of the current study was to determine the function of VEGF during testis development in bulls. We used an explant tissue culture and treatment approach to test the hypothesis that VEGFA-164 could regulate the biological activity of bovine germ cells. We demonstrate that VEGFA, KDR, and FLT1 proteins are expressed in germ and somatic cells in the bovine testis. Treatment of bovine testis tissue with VEGFA in vitro resulted in significantly more germ cells following 5 days of culture when compared with controls. Quantitative real-time RT-PCR analysis determined that VEGF treatment stimulated an intracellular response that prevents germ cell death in bovine testis tissue explants, as indicated by increased expression of BCL2 relative to BAX and decreased expression of BNIP3 at 3, 6, and 24 h during culture. Blocking VEGF activity in vitro using antisera against KDR and VEGF significantly reduced the number of germ cells in VEGF-treated testis tissue to control levels at 120 h. Testis grafting provided in vivo evidence that bovine testis tissue treated with VEGFA for 5 days in culture contained significantly more differentiating germ cells compared with controls. These findings support the conclusion that VEGF supports germ cell survival and sperm production in bulls.

Spermatogonial stem cell transplantation, testicular function, and restoration of male fertility in mice.

Mammalian spermatogonial stem cells, sometimes called male germline stem cells, are a small population of adult tissue-specific stem cells present in the testis. Formation of the spermatogonial stem cell population early in life and differentiation of spermatogonial stem cells in adults are responsible for continual production of sperm in the testis. Unfortunately, there are no specific biochemical or morphological markers for spermatogonial stem cells, so investigation of this cell type requires specific and consistent approaches to ensure valid data are obtained. Currently, the only assay for the presence of spermatogonial stem cells in a cell suspension is the spermatogonial stem cell transplantation technique. This requires the transfer of cells from a donor animal into the testis of a recipient animal, in which the spermatogonial stem cells will colonize and initiate donor-derived spermatogenesis. Although there is no specific marker for spermatogonial stem cells, several cell surface markers have been used to enrich for these cells prior to transplantation. Thus, selection and transplantation of spermatogonial stem cells can be used to investigate basic mechanisms regulating them. Successful transplantation and donor-derived spermatogenesis in recipient animals can lead to the restoration of fertility in infertile males. In combination with spermatogonial stem cell culture, this transplantation technique can also be used for the purpose of generating transgenic animals through the male germline. This chapter describes the methods for spermatogonial stem cell transplantation and how this approach is used to investigate testicular function.

Potential functions of retinoic acid receptor A in Sertoli cells and germ cells during spermatogenesis.

Elucidation of the retinoid signaling circuitry in the testis is critical to understanding how male germ cells develop to spermatozoa. Retinoic acid receptor A protein (RARA) is an essential mediator of retinoid signaling in the testis, as shown by a sterility phenotype observed for retinoic acid receptor A gene (Rara) knockout male mice. The seminiferous tubules of Rara knockout mice showed varying degrees of germ-cell degeneration. A dramatic increase in apoptosis of early meiotic prophase spermatocytes was observed in these tubules compared to the wild-type tubules. Germ-cell loss was dependent on the stages of the spermatogenic cycle: germ-cell loss was negligible in stages I-V, but severe after stages VIII and IX of the spermatogenic cycle. Using spermatogonial transplantation, the individual function of RARA in Sertoli cells or germ cells was determined. The wild-type donor germ cells, transplanted into Rara knockout testes, colonized and proliferated in the RARA-deficient microenvironment. The donor-derived cells were mostly early meiotic prophase spermatocytes, with few more advanced germ cells detected. Conversely, when Rara-deficient germ cells were injected into the microenvironment that express RARA, establishment of donor-derived germ-cell colonies was rare, but remarkably, once colonized, Rara-deficient germ cells progressed normally through spermatogenesis. These results together suggest that RARA may function in Sertoli cells to promote the survival and development of early meiotic prophase spermatocytes, whereas RARA in germ cells functions to increase the proliferation and differentiation of spermatogonia, prior to meiotic prophase.

Analysis of gene expression in bovine testis tissue prior to ectopic testis tissue xenografting and during the grafting period.

The purpose of this study was to identify factors that contribute to bovine testis development and donor age-dependent differences in the abilities of bovine ectopic testis tissue grafts to produce elongated spermatids. We used real-time RT-PCR and microarrays to evaluate and to identify the expression of genes that are involved in Sertoli and germ cell development in bovine testis tissues. Testis tissues were obtained from 2-, 4-, and 8-wk-old bull calves and were grafted immediately. Grafted bovine testis tissue was removed from mice, RNA was isolated from the grafts, and real-time RT-PCR was used to evaluate gene expression during the grafting period. In addition, the gene expression in the donor tissue was analyzed using Affymetrix Bovine GeneChips, to identify differentially expressed genes. Examination of the testis tissue grafts indicated that Sertoli cell-specific gene expression was lower in 8-wk donor tissue grafts compared to the donors of other ages. Furthermore, the expression of KIT, which is a germ cell-specific gene, was low in testis tissue grafts. Microarray analysis of the donor tissue showed that several genes that are involved in angiogenesis or tissue growth were differentially expressed in 2-, 4-, and 8-wk-old bovine testes. The levels of expression of the genes for angiogenin, transgelin, thrombomodulin, early growth response 1, insulin-like growth factor 2, and insulin-like growth factor-binding protein 3 were lower in testis tissues from older animals. Using these data, it will be possible in the future to manipulate the testis xenograft microenvironment so as to improve the efficiency of sperm production within the graft.