Feeding spent hemp biomass to lactating dairy cows: Effects on performance, milk components and quality, blood parameters, and nitrogen metabolism.

The legalization of industrial hemp by the 2018 Farm Bill in the United States has driven a sharp increase in its cultivation, including for cannabinoid extraction. Spent hemp biomass (SHB), produced from the extraction of cannabinoids, can potentially be used as feed for dairy cows; however, it is still illegal to do so in the United States, according to the US Food and Drug Administration Center for Veterinary Medicine, due to the presence of cannabinoids and the lack of data on the effect on animals. To assess the safety of this byproduct as feed for dairy cows, late-lactation Jersey cows (245 ± 37 d in milk; 483 ± 38 kg body weight; 10 multiparous and 8 primiparous) received a basal total mixed ration (TMR) diet plus 13% alfalfa pellet (CON) or 13% pelleted SHB for 4 wk (intervention period [IP]) followed by 4 wk of withdrawal period (WP), where all cows received only the basal TMR during WP. The dry matter intake (DMI), body weight, body condition score, milk yield, milk components, and fatty acid profile, blood parameters, N metabolism, methane emission, and activity were measured. Results indicated that feeding SHB decreased DMI mainly due to the low palatability of the SHB pellet, as the cows consumed only 7.4% of the total TMR with 13.0% SHB pellet offered in the ration. However, milk yield was not affected during the IP and was higher than CON during the WP, leading to higher milk yield/DMI. Milk components were not affected, except for a tendency in decreased fat percentage. Milk fat produced by cows fed SHB had a higher proportion of oleate and bacteria-derived fatty acids than CON. The activity of the cows was not affected, except for a shorter overall lying time in SHB versus CON cows during the IP. Blood parameters related to immune function were not affected. Compared with CON, cows fed SHB had a lower cholesterol concentration during the whole experiment and higher ß-hydroxybutyric acid during the WP, while a likely low-grade inflammation during the IP was indicated by higher ceruloplasmin and reactive oxidative metabolites. Other parameters related to liver health and inflammatory response were unaffected, except for a tendency for higher activity of alkaline phosphatase during IP and a lower activity of gamma-glutamyl transferase during WP in the SHB group versus CON. The bilirubin concentration was increased in cows fed SHB, suggesting a possible decrease in the clearance ability of the liver. Digestibility of the dry matter and protein and methane emission were not affected by feeding SHB. The urea, purine derivatives, and creatinine concentration in urine was unaffected, but cows fed SHB had higher N use efficiency and lower urine volume. Altogether, our data revealed a relatively low palatability of SHB affecting DMI with minimal biological effects, except for a likely low-grade inflammation, a higher N use efficiency, and a possible decrease in liver clearance. Overall, the data support the use of SHB as a safe feed ingredient for lactating dairy cows.

Effect of Fetal Pituitary-Testes Suppression on Brain Sexual Differentiation and Reproductive Function in Male Sheep.

We previously demonstrated that treating fetal lambs on gestational day 62 with the long-acting gonadotrophin-releasing hormone (GnRH) antagonist degarelix (DG) suppresses pituitary-testicular function during midgestation. The objective of this study was to investigate whether impaired gonadotrophic drive during this fetal period has enduring effects on sexual differentiation and reproductive function in adult male sheep. We assessed the effects of prenatal administration of DG, with or without testosterone (T) replacement, on various sexually dimorphic behavioral traits in adult rams, including sexual partner preferences, as well as neuroendocrine responsiveness and testicular function. Our findings revealed that DG treatment had no effect on genital differentiation or somatic growth. There were some indications that DG treatment suppressed juvenile play behavior and adult sexual motivation; however, male-typical sexual differentiation of reproductive behavior, sexual partner preference, and gonadotropin feedback remained unaffected and appeared to be fully masculinized and defeminized. DG-treated rams showed an increased LH response to GnRH stimulation and a decreased T response to human chorionic gonadotropin stimulation, suggesting impaired Leydig cell function and reduced T feedback. Both effects were reversed by cotreatment with T propionate. DG treatment also suppressed the expression of CYP17 messenger RNA, a key enzyme for T biosynthesis. Despite the mild hypogonadism induced by DG treatment, ejaculate volume, sperm motility, and sperm morphology were not affected. In summary, these results suggest that blocking GnRH during midgestation does not have enduring effects on brain sexual differentiation but does negatively affect the testes' capacity to synthesize T.

Peroxisome Proliferator-Activated Receptor Activation in Precision-Cut Bovine Liver Slices Reveals Novel Putative PPAR Targets in Periparturient Dairy Cows.

Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the Peroxisome Proliferator-activated Receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is time consuming, resource intensive, and prone to errors, with the resulting cells losing characteristic phenotypical traits within hours. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Three primiparous Jersey cows were enrolled in the experiment, and PCLS from each were prepared prepartum (-8.0 ± 3.6 DIM) and postpartum (+7.7± 1.2 DIM) and treated independently with a variety of PPAR agonists and antagonists: the PPARα agonist WY-14643 and antagonist GW-6471; the PPARδ agonist GW-50156 and antagonist GSK-3787; and the PPARγ agonist rosiglitazone and antagonist GW-9662. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triacylglycerol (TAG) concentration was measured. PCLS obtained from postpartum cows and treated with a PPARγ agonist displayed upregulation of ACADVL and LIPC while those treated with PPARδ agonist had increased expression of LIPC, PPARD, and PDK4. In PCLS from prepartum cows, transcription of LIPC was increased by all PPAR agonists and NEFA. TAG concentration tended to be larger in tissue slices treated with PPARδ agonist compared to CTR. Use of PPAR isotype-specific antagonists in PCLS cultivated in autologous blood serum failed to decrease expression of PPAR targets, except for PDK4, which was confirmed to be a PPARδ target. Transcriptome sequencing revealed considerable differences in response to PPAR agonists at a false discovery rate-adjusted p-value of 0.2, with the most notable effects exerted by the PPARδ and PPARγ agonists. Differentially expressed genes were mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly available dataset of predicted PPAR target genes, and supplementing our findings with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the periparturient dairy cow.

Increasing vitamin D levels to improve fertilization rates in cattle.

Recently, interest in supplementing vitamin D (Vit D) to improve aspects of health, mainly in human fertility, has emerged. Still, supplementation of Vit D above the minimum required levels has yet to be explored in cattle despite evidence for Vit D receptors in reproductive tissues. The objective of this study was to establish if a dose-response relationship exists between Vit D exposure and success of in vitro production (IVP) of embryos and, if acute supplementation of Vit D improves pregnancy rates during timed artificial insemination (TAI) of dairy cows. Cumulus-oocyte complexes (COCs) were obtained from ovaries acquired from a local abattoir and cultured in five different IVP treatments from three separate collections (Control, 50, 100, 150, and 200 ng/mL of 1,25(OH)2D3; n = 20-30 COCs/group). In Experiment 2, dairy breed cows (n = 100) were synchronized for TAI with the PresynchOvsynch protocol. Cows received 150,000 IU of Vit D (n = 48) or castor oil as control (n = 53) along with gonadotropin-releasing hormone (GnRH) 24 h before TAI. Serum samples were collected before and 24 h after treatment. A small cohort of cows (n = 4) received the same treatments in two separate cycles and follicular fluid (FF) was collected after 24 h for calcidiol (25OHD) analyses. Increased concentrations of Vit D resulted in decreased rates of maturation of COC (150 and 200 ng/mL vs. control and 50 ng/mL; P = 0.01). Supplementation with 50 ng/mL resulted in greater numbers of early blastocyst and blastocyst stage embryos (P < 0.009). Pregnancy at first breeding did not differ (P = 0.13) between groups, but serum 25OHD increased in treated females after 24 h (P = 0.002). The FF 25OHD levels were reflective of serum levels, however, the observed increase in the treatment cycle (P = 0.04) was parallel to an overall increase in serum 25OHD during the entire second cycle, likely due to increased environmental sunlight exposure (March, control vs. May, treatment). A similar increase in the serum 25OHD in the lactating commercial herd maintained in covered housing was not observed, although experiments were conducted during a similar timeframe. This herd had levels of 25OHD near the low end of sufficiency according to National Research Council (NRC) guidelines. We conclude mild Vitamin D supplementation with concentrations at the higher end of NRC guidelines can improve maturation rates of recovered COCs. However, longer term supplementation may be needed to appreciate any benefits on fertility.

Vitamin D is an important hormone that among other things, contributes to bone health, immunity, and reproduction. Recently, research has linked vitamin D to fertility in other species (primates), and therefore the objectives of the current research were to determine if mild supplementation with Vitamin D impacted fertility in female cattle. A dose-dependent relationship was detected between concentrations of vitamin D and embryo development. The concentration of 50 ng/mL of vitamin D appeared to be beneficial to early embryogenesis. Studies in dairy-breed females indicated serum levels of vitamin D correlated well with intrafollicular levels in the periovulatory follicle. Finally, a fertility trial investigated if a single dose of vitamin D improves fertility when administered before artificial insemination in cattle. There were no detectable benefits to this brief supplementation with vitamin D on measures of fertility in this group. It is concluded supplementation with vitamin D improves embryo development in vitro, but brief supplementation did not impact pregnancy success. Longer-term supplementation with vitamin D may be needed to appreciate any measurable benefits on fertility.

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses.

The specific role of gonadotropin-releasing hormone (GnRH) on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma luteinizing hormone (LH) and T levels. In addition, we measured the effect of degarelix-treatment on messenger RNA (mRNA) expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHß-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.

Effects of Deoxynivalenol and Fumonisins Fed in Combination to Beef Cattle: Immunotoxicity and Gene Expression.

We evaluated the effects of a treatment diet contaminated with 1.7 mg deoxynivalenol and 3.5 mg fumonisins (B1, B2 and B3) per kg ration on immune status and peripheral blood gene expression profiles in finishing-stage Angus steers. The mycotoxin treatment diet was fed for a period of 21 days followed by a two-week washout period during which time all animals consumed the control diet. Whole-blood leukocyte differentials were performed weekly throughout the experimental and washout period. Comparative profiles of CD4+ and CD8+ T cells, along with bactericidal capacity of circulating neutrophils and monocytes were evaluated at 0, 7, 14, 21 and 35 days. Peripheral blood gene expression was measured at 0, 7, 21 and 35 days via RNA sequencing. Significant increases in the percentage of CD4-CD8+ T cells were observed in treatment-fed steers after two weeks of treatment and were associated with decreased CD4:CD8 T-cell ratios at this same timepoint (p ≤ 0.10). No significant differences were observed as an effect of treatment in terms of bactericidal capacity at any timepoint. Dietary treatments induced major changes in transcripts associated with endocrine, metabolic and infectious diseases; protein digestion and absorption; and environmental information processing (inhibition of signaling and processing), as evaluated by dynamic impact analysis. DAVID analysis also suggested treatment effects on oxygen transport, extra-cellular signaling, cell membrane structure and immune system function. These results indicate that finishing-stage beef cattle are susceptible to the immunotoxic and transcript-inhibitory effects of deoxynivalenol and fumonisins at levels which may be realistically encountered in feedlot situations.

Feeding selenium-biofortified alfalfa hay during the preconditioning period improves growth, carcass weight, and nasal microbial diversity of beef calves.

We previously reported that feeding Se-biofortified alfalfa hay to weaned beef calves in a preconditioning program decreases morbidity and mortality during the feedlot period. To understand the mode of action by which supranutritional Se supplementation supports calf health, we examined the effect of agronomic Se-biofortification on nasal microbiome and fecal parasites. Recently weaned Angus-cross beef calves (n = 30) were randomly assigned to two groups and fed an alfalfa hay-based diet for 9 weeks in a preconditioning program. Alfalfa hay was harvested from fields fertilized with sodium selenate at a rate of 0 or 90 g Se/ha. Calculated Se intake from dietary sources was 1.09 and 27.45 mg Se/calf per day for calves consuming alfalfa hay with Se concentrations of 0.06 and 3.47 mg Se/kg dry matter, respectively. Feeding Se-biofortified alfalfa hay for 9 weeks was effective at increasing whole-blood Se concentrations (556 ± 11 vs 140 ± 11 ng/mL; P < 0.001) and increasing body weight (PTreatment, = 0.03) in weaned beef calves. Slaughter yield grades were higher for calves that had been fed Se-enriched alfalfa hay during the preconditioning period (PTreatment = 0.008). No significant differences were observed in fecal parasite load, which remained low. The nasal microbiome and microbiota diversity within calves and across calves expanded from weaning (week 0) to the feedlot period (week 12), which was promoted by feeding Se-biofortified alfalfa hay. Especially concerning was the expansion of nasal Mycoplasmataceae in the feedlot, which reached over 50% of the total microbiota in some calves. In conclusion, we identified dietary Se-biofortified alfalfa hay as a potential promoter of nasal microbiome genome and microbiota diversity, which may explain in part high-Se benefits for prevention of bovine respiratory disease complex in beef calves.

Selenium biofortified alfalfa hay fed in low quantities improves selenium status and glutathione peroxidase activity in transition dairy cows and their calves.

The hypothesis of the study was that feeding a relatively low amount of Se biofortified alfalfa hay during the dry period and early lactation would improve selenium status and glutathione peroxidase activity in dairy cows and their calves. Ten Jersey and 8 Holstein primiparous dairy cows were supplemented with Se biofortified (TRT; n = 9) or non-biofortified (CTR; n = 9) alfalfa hay at a rate of 1 kg/100 kg of BW mixed with the TMR from 40 d prior parturition to 2 weeks post-partum. Se concentration in whole blood, liver, milk, and colostrum, the transfer of Se to calves, and the glutathione peroxidase (GPx) activity were assessed. TRT had 2-fold larger (P < 0.05) Se in blood v. CTR that resulted in larger Se in liver and colostrum but not milk and larger GPx activity in plasma and erythrocytes but not in milk. Compared to CTR, calves from TRT had larger Se in blood but only a numerical (P = 0.09) larger GPx activity in plasma. A positive correlation was detected between Se in the blood and GPx activity in erythrocytes and plasma in cows. Our results demonstrated that feeding pregnant primiparous dairy cows with a relatively low amount of Se-biofortified alfalfa hay is an effective way to increase Se in the blood and liver, leading to greater antioxidant activity via GPx. The same treatment was effective in improving Se concentration in calves but had a modest effect on their GPx activity. Feeding Se biofortified hay increased Se concentration in colostrum but not in milk.

Role for Kisspeptin and Neurokinin B in Regulation of Luteinizing Hormone and Testosterone Secretion in the Fetal Sheep.

Evidence suggests that the hypothalamic-pituitary-gonadal (HPG) axis is active during the critical period for sexual differentiation of the ovine sexually dimorphic nucleus, which occurs between gestational day (GD) 60 and 90. Two possible neuropeptides that could activate the fetal HPG axis are kisspeptin and neurokinin B (NKB). We used GD85 fetal lambs to determine whether intravenous administration of kisspeptin-10 (KP-10) or senktide (NKB agonist) could elicit luteinizing hormone (LH) release. Immunohistochemistry and fluorescent in situ hybridization (FISH) were employed to localize these peptides in brains of GD60 and GD85 lamb fetuses. In anesthetized fetuses, KP-10 elicited robust release of LH that was accompanied by a delayed rise in serum testosterone in males. Pretreatment with the GnRH receptor antagonist (acyline) abolished the LH response to KP-10, confirming a hypothalamic site of action. In unanesthetized fetuses, senktide, as well as KP-10, elicited LH release. The senktide response of females was greater than that of males, indicating a difference in NKB sensitivity between sexes. Gonadotropin-releasing hormone also induced a greater LH discharge in females than in males, indicating that testosterone negative feedback is mediated through pituitary gonadotrophs. Kisspeptin and NKB immunoreactive cells in the arcuate nucleus were more abundant in females than in males. Greater than 85% of arcuate kisspeptin cells costained for NKB. FISH revealed that the majority of these were kisspeptin/NKB/dynorphin (KNDy) neurons. These results support the hypothesis that kisspeptin-GnRH signaling regulates the reproductive axis of the ovine fetus during the prenatal critical period acting to maintain a stable androgen milieu necessary for brain masculinization.

2,4-Thiazolidinedione in Well-Fed Lactating Dairy Goats: I. Effect on Adiposity and Milk Fat Synthesis.

Background: In a prior experiment, treatment of goats with the putative PPARγ agonist 2,4-thiazolidinedione (2,4-TZD) did not affect milk fat or expression of milk-fat related genes. The lack of response was possibly due to deficiency of vitamin A and/or a poor body condition of the animals. In the present experiment, we tested the hypothesis that PPARγ activation affects milk fat synthesis in goats with a good body condition and receiving adequate levels of vitamin A. Methods: Lactating goats receiving a diet that met NRC requirements, including vitamin A, were injected with 8 mg/kg BW of 2,4-TZD (n = 6) or saline (n = 6; CTR) daily for 26 days. Blood metabolic profiling and milk yield and components were measured including fatty acid profile. Expression of genes related to glucose and lipid metabolism was measured in adipose tissue and in mammary epithelial cells (MEC). Size of adipocytes was assessed by histological analysis. Results: NEFA, BHBA, and fatty acids available in plasma decreased while glucose increased in 2,4-TZD vs. CTR. Size of cells and expression of insulin signaling and glucose metabolism-related genes were larger in 2,4-TZD vs. CTR in adipose tissue. In MEC, expression of SCD1 and desaturation of stearate was lower in 2,4-TZD vs. CTR. Conclusions: Overall data revealed a lack of PPARγ activation by 2,4-TZD and no effect on milk fat synthesis despite a strong anti-lipolysis effect on adipose tissue.

2,4-Thiazolidinedione Treatment Improves the Innate Immune Response in Dairy Goats with Induced Subclinical Mastitis.

Mastitis is a major disease in dairy cows resulting in significant economic losses. In vitro works suggest that ruminants peroxisome proliferator-activated receptor gamma (PPARγ) can aid in improving the response to mastitis and can control milk fat synthesis. The objectives of the present experiment were to test if treatment with the putative PPARγ agonist 2,4-thiazolidinedione (TZD) improves (1) the response to subclinical mastitis and (2) milk fat production. Lactating goats received daily injections of 8 mg/kg BW of TZD or saline for 3 weeks. After one week of TZD injection, half of the goats in each group received intramammary infusion of Strep. uberis or saline in both halves for a total of 4 groups (n = 6/group). TZD treatment did not affect milk fat but had positive effect on milk somatic cells count, blood nonesterified fatty acids, inflammatory markers, and liver function. TZD significantly increased myeloperoxidase but did not affect leukocytes phagocytosis or insulin. TZD increased adipocytes size and had minor effect on expression of PPARγ target genes in mammary epithelial cells but not in adipose tissue. Overall, TZD ameliorated the response to intramammary infection but the effect on milk fat synthesis and expression of related transcripts was less than expected.

Weaned beef calves fed selenium-biofortified alfalfa hay have an enriched nasal microbiota compared with healthy controls.

Selenium (Se) is an essential trace mineral important for immune function and overall health of cattle. The nasopharyngeal microbiota in cattle plays an important role in overall respiratory health, especially when stresses associated with weaning, transport, and adaptation to a feedlot affect the normal respiratory defenses. Recent evidence suggests that cattle diagnosed with bovine respiratory disease complex have significantly less bacterial diversity. The objective of this study was to determine whether feeding weaned beef calves Se-enriched alfalfa (Medicago sativa) hay for 9 weeks in a preconditioning program prior to entering the feedlot alters nasal microbiota. Recently weaned beef calves (n = 45) were blocked by sex and body weight, randomly assigned to 3 treatment groups with 3 pens of 5 calves per treatment group, and fed an alfalfa hay based diet for 9 weeks. Alfalfa hay was harvested from fields fertilized with sodium selenate at a rate of 0, 45.0 or 89.9 g Se/ha. Blood samples were collected biweekly and analyzed for whole-blood Se concentrations. Nasal swabs were collected during week 9 from one or two calves from each pen (total n = 16). Calculated Se intake from dietary sources was 3.0, 15.6, and 32.2 mg Se/head/day for calves consuming alfalfa hay with Se concentrations of 0.34 to 2.42 and 5.17 mg Se/kg dry matter, respectively. Whole-blood Se concentrations after 8 weeks of feeding Se-fertilized alfalfa hay were dependent upon Se-application rates (0, 45.0, or 89.9 g Se/ha) and were 155, 345, and 504 ng/mL (PLinear < 0.0001). Microbial DNA was extracted from nasal swabs and amplified and sequenced. Alpha rarefaction curves comparing the species richness (observed OTUs) and overall diversity (Chao1, Observed OTU, and Shannon index) between calves fed selenium-biofortified alfalfa hay compared with control calves showed that Se-supplementation tended to be associated with an enriched nasal microbiota. ANOSIM of unweighted UniFrac distances showed that calves fed high Se-biofortified alfalfa hay clustered separately when compared with control calves in the PCoA plot (R = 0.216, P = 0.04). The bacterial orders Lactobacillales and Flavobacteriales were increased in healthy control calves compared with Clostridiales and Bacteroidales being increased in calves fed Se-biofortified alfalfa hay. Although there were strong trends, no significant differences were noted for any of the bacterial taxa. Based upon these findings, we suggest that weaned beef calves fed Se-biofortified hay tend to have an enriched nasal microbiota. Feeding Se-biofortified alfalfa hay to weaned beef calves prior to entering the feedlot is a strategy for increasing nasopharyngeal microbial diversity.

Excess Testosterone Exposure Alters Hypothalamic-Pituitary-Testicular Axis Dynamics and Gene Expression in Sheep Fetuses.

Prenatal exposure to excess androgen may result in impaired adult fertility in a variety of mammalian species. However, little is known about what feedback mechanisms regulate gonadotropin secretion during early gestation and how they respond to excess T exposure. The objective of this study was to determine the effect of exogenous exposure to T on key genes that regulate gonadotropin and GnRH secretion in fetal male lambs as compared with female cohorts. We found that biweekly maternal testosterone propionate (100 mg) treatment administered from day 30 to day 58 of gestation acutely decreased (P < .05) serum LH concentrations and reduced the expression of gonadotropin subunit mRNA in both sexes and the levels of GnRH receptor mRNA in males. These results are consistent with enhanced negative feedback at the level of the pituitary and were accompanied by reduced mRNA levels for testicular steroidogenic enzymes, suggesting that Leydig cell function was also suppressed. The expression of kisspeptin 1 mRNA, a key regulator of GnRH neurons, was significantly greater (P < .01) in control females than in males and reduced (P < .001) in females by T exposure, indicating that hypothalamic regulation of gonadotropin secretion was also affected by androgen exposure. Although endocrine homeostasis was reestablished 2 weeks after maternal testosterone propionate treatment ceased, additional differences in the gene expression of GnRH, estrogen receptor-ß, and kisspeptin receptor (G protein coupled receptor 54) emerged between the treatment cohorts. These changes suggest the normal trajectory of hypothalamic-pituitary axis development was disrupted, which may, in turn, contribute to negative effects on fertility later in life.

Effect of Testosterone on Neuronal Morphology and Neuritic Growth of Fetal Lamb Hypothalamus-Preoptic Area and Cerebral Cortex in Primary Culture.

Testosterone plays an essential role in sexual differentiation of the male sheep brain. The ovine sexually dimorphic nucleus (oSDN), is 2 to 3 times larger in males than in females, and this sex difference is under the control of testosterone. The effect of testosterone on oSDN volume may result from enhanced expansion of soma areas and/or dendritic fields. To test this hypothesis, cells derived from the hypothalamus-preoptic area (HPOA) and cerebral cortex (CTX) of lamb fetuses were grown in primary culture to examine the direct morphological effects of testosterone on these cellular components. We found that within two days of plating, neurons derived from both the HPOA and CTX extend neuritic processes and express androgen receptors and aromatase immunoreactivity. Both treated and control neurites continue to grow and branch with increasing time in culture. Treatment with testosterone (10 nM) for 3 days significantly (P < 0.05) increased both total neurite outgrowth (35%) and soma size (8%) in the HPOA and outgrowth (21%) and number of branch points (33%) in the CTX. These findings indicate that testosterone-induced somal enlargement and neurite outgrowth in fetal lamb neurons may contribute to the development of a fully masculine sheep brain.

Prenatal influence of an androgen agonist and antagonist on the differentiation of the ovine sexually dimorphic nucleus in male and female lamb fetuses.

The ovine sexually dimorphic nucleus (oSDN) is 2 times larger in rams than in ewes. Sexual differentiation of the oSDN is produced by testosterone exposure during the critical period occurring between gestational day (GD)60 and GD90 (term, 147 d). We tested the hypothesis that testosterone acts through the androgen receptor to control development of the male-typical oSDN. In experiment 1, pregnant ewes received injections of vehicle, androgen receptor antagonist flutamide, or nonaromatizable androgen dihydrotestosterone (DHT) propionate during the critical period. Fetuses were delivered at GD135. Both antagonist and agonist treatments significantly reduced mean oSDN volume in males but had no effects in females. Experiment 2, we analyzed the effect of treatments on the fetal hypothalamic-pituitary-gonadal axis to determine whether compensatory changes in hormone secretion occurred that could explain the effect of DHT. Pregnant ewes were injected with vehicle, flutamide, or DHT propionate from GD60 to GD84, and fetuses were delivered on GD85. Flutamide significantly increased LH and testosterone in males, whereas DHT significantly decreased both hormones. In females, LH was unaffected by flutamide but significantly reduced by DHT exposure. DHT significantly decreased pituitary gonadotropin and hypothalamic kisspeptin mRNA expression in males and females. These results suggest that androgen receptor mediates the effect of testosterone on oSDN masculinization, because this process was blocked by the androgen receptor antagonist flutamide in eugonadal males. In contrast, the reduction of oSDN volume observed after DHT exposure appears to be mediated by a negative feedback mechanism exerted on the hypothalamus to reduce LH and testosterone secretion. The reduced androgen exposure most likely accounted for the decreased oSDN volume. We conclude that, during the critical period, the male reproductive axis in long gestation species, such as sheep, is sufficiently developed to react to perturbations in serum androgens and mitigate disruptions in brain masculinization.

Effect of supranutritional maternal and colostral selenium supplementation on passive absorption of immunoglobulin G in selenium-replete dairy calves.

Selenium (Se) is an essential micronutrient for ruminant animals affecting both performance and immune functions. Adding 3 mg of Se/L (in the form of Na selenite) to colostrum has been shown to improve IgG absorption in Se-deficient newborn dairy calves. The objective of our study was to determine the effect of supranutritional maternal and colostral Se supplementation on IgG status of Se-replete dairy calves. The study design was a 2 × 2 × 2 factorial design. During the last 8 wk before calving, dairy cows at a commercial dairy were fed either 0 (control cows) or 105 mg of Se-yeast once weekly (supranutritional Se-yeast-supplemented cows), in addition to Na selenite at 0.3 mg of Se/kg of DM in their ration. After birth, calves were fed pooled colostrum from control or supranutritional Se-yeast-supplemented cows to which 0 or 3 mg of Se/L (in the form of Na selenite) was added. Concentrations of whole-blood (WB) Se and serum Se measured at birth and at 48 h and 14 d of age, and serum IgG concentrations measured at 48 h and 14 and 60 d of age were determined. Calves born to Se-yeast-supplemented cows had higher WB-Se and serum-Se concentrations for the first 2 wk, and higher IgG absorption efficiency (62% at 48 h), resulting in higher serum-IgG concentrations (43% at 48 h and 65% at 14 d) and higher total serum-IgG content (50% at 48 h and 75% at 14 d), compared with calves born to control cows. Calves that received colostrum with added Na selenite had higher WB-Se concentrations for the first 2 wk, but only at 14 d of age were serum-Se concentrations, serum-IgG concentrations (53% higher), and total serum-IgG content (56% higher) higher, compared with calves that were fed colostrum without added Na selenite. Calves born to Se-yeast-supplemented cows that received colostrum from Se-yeast cows without added Na selenite had a higher IgG absorption efficiency compared with all other treatment groups. Our results support that feeding cows supranutritional Se-yeast supplement during the dry period or spiking colostrum with Na selenite both improve IgG status of Se-replete calves.

Cell death in the central division of the medial preoptic nucleus of male and female lamb fetuses.

The medial preoptic area of the adult sheep contains an ovine sexually dimorphic nucleus (oSDN) that is larger and has more neurons in males than in females. In the lamb fetus, the nascent oSDN occupies the central division of the medial preoptic nucleus (MPNc) and consists of a cluster of cells that is organized by the action of testosterone during gestational days 60-90 of a 147 day term pregnancy. The current study sought to determine whether programmed cell death contributes to the emergence of the oSDN. Male and female lamb fetuses were euthanized at different ages spanning the period during which the oSDN is organized. The expression of the pro- and anti-apoptotic genes bcl-2 and bax, respectively, was measured by quantitative RT-PCR to assess possible sex differences in neuron vulnerability to programmed cell death. The appearance of DNA-fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and used to estimate the occurrence of apoptotic cell death. We found that bcl-2 and bax mRNA expression in the medial preoptic area of the developing lamb fetus decreased during the last half of the 147-day gestation. The ratio of bcl-2/bax gene expression was highest at gestational day 85 but was equivalent between males and females. TUNEL staining in the MPNc was very low and although it decreased significantly with age, it was not significantly different between sexes. These results using two different methods to assess cell death indicate that a sex difference in the incidence of cell death is not a primary mechanism leading to sexual differentiation of the oSDN.

Effect of feeding selenium-fertilized alfalfa hay on performance of weaned beef calves.

Selenium (Se) is an essential micronutrient in cattle, and Se-deficiency can affect morbidity and mortality. Calves may have greater Se requirements during periods of stress, such as during the transitional period between weaning and movement to a feedlot. Previously, we showed that feeding Se-fertilized forage increases whole-blood (WB) Se concentrations in mature beef cows. Our current objective was to test whether feeding Se-fertilized forage increases WB-Se concentrations and performance in weaned beef calves. Recently weaned beef calves (n = 60) were blocked by body weight, randomly assigned to 4 groups, and fed an alfalfa hay based diet for 7 wk, which was harvested from fields fertilized with sodium-selenate at a rate of 0, 22.5, 45.0, or 89.9 g Se/ha. Blood samples were collected weekly and analyzed for WB-Se concentrations. Body weight and health status of calves were monitored during the 7-wk feeding trial. Increasing application rates of Se fertilizer resulted in increased alfalfa hay Se content for that cutting of alfalfa (0.07, 0.95, 1.55, 3.26 mg Se/kg dry matter for Se application rates of 0, 22.5, 45.0, or 89.9 g Se/ha, respectively). Feeding Se-fertilized alfalfa hay during the 7-wk preconditioning period increased WB-Se concentrations (P Linear<0.001) and body weights (P Linear = 0.002) depending upon the Se-application rate. Based upon our results we suggest that soil-Se fertilization is a potential management tool to improve Se-status and performance in weaned calves in areas with low soil-Se concentrations.

ESI-Mass spectrometric and HPLC elucidation of a new ergot alkaloid from perennial ryegrass hay silage associated with bovine reproductive problems.

This case report involves four dairies in the Willamette Valley, Oregon, which experienced reproductive problems associated with the presence of a large, previously unidentified, peak eluting at 5 min in a standard ergovaline high-performance liquid chromatography assay of perennial ryegrass silage fed to those animals. Mycotoxin analysis of the silage was negative, as was serological screening of the herds for infectious bovine rhinotracheitis, bovine diarrhea virus and Leptospirosis, including culturing of urine for Leptospira hardjo hardjobovis. Prolactin concentrations were low in most cattle, consistent with ingestion of ergot alkaloids. We believe that this peak represents a novel ergot alkaloid-related compound due to its extractability with Ergosil, its detectability due to fluorescence, and its chromatographic retention between ergovaline (mw = 533) and ergotamine (mw = 581). Its molecular weight was calculated as 570 owing to the predominance of a m/z 593.5 ion in the full scan ESI(+)MS and its deduced tendency to complex with Na(+) (as m/z 593) or K(+) (as m/z 609) ions. We offer rationales for elucidation of the structure of this compound, with the closest starting point comprising an m.w. of 566-a fructofuranosyl-(2-1)-O-beta-D-fructofuranoside derivative of 6,7-secoergoline from Claviceps fusiformis. This m.w. requires modifications, such as reduction of two double bonds in the secoergoline component to give the target 570 m.w. Despite the lack of a definitive structure, the analysis herein provides a starting point for eventual elucidation of this apparently new ergot alkaloid, and to guide and encourage further investigation as to its association with endophyte toxicosis in livestock.

Separate critical periods exist for testosterone-induced differentiation of the brain and genitals in sheep.

Sheep exposed to testosterone during a critical period from gestational day (GD) 30 to GD 90 develop masculine genitals and an enlarged male-typical ovine sexually dimorphic nucleus of the preoptic area (oSDN). The present study tested the hypothesis that separate critical periods exist for masculinization of these two anatomical end points. Pregnant ewes were treated with testosterone propionate (TP) either from GD 30 to GD 60 (early TP) or GD 60 to GD 90 (late TP). Control (C) pregnant ewes were treated with corn oil. Fetuses were delivered at GD 135 and the volume of the oSDN was measured. Early TP females possessed a penis and a scrotum devoid of testes, whereas late TP and C females had normal female genitals. Neither period of TP exposure grossly affected the genitals of male fetuses. Despite masculinized genitals, the mean volume of the oSDN in early TP females (0.32 ± 0.06 mm³) was not different from C females (0.24 ± 0.02 mm³) but was significantly enlarged in late TP females (0.49 ± 0.04 mm³; P < 0.05 vs. C) when the genitals appeared normal. In contrast, the volume of the oSDN in late TP males (0.51 ± 0.02 mm³) was not different from C males (0.51 ± 0.04 mm³) but was significantly smaller in the early TP males (0.35 ± 0.04 mm³; P < 0.05 vs. C). These results demonstrate that the prenatal critical period for androgen-dependent differentiation of the oSDN occurs later than, and can be separated temporally from, the period for development of masculine genitals.