Early prenatal androgen exposure reduces testes size and sperm concentration in sheep without altering neuroendocrine differentiation and masculine sexual behavior.

Prenatal androgens are largely responsible for growth and differentiation of the genital tract and testis and for organization of the control mechanisms regulating male reproductive physiology and behavior. The aim of the present study was to evaluate the impact of inappropriate exposure to excess testosterone (T) during the first trimester of fetal development on the reproductive function, sexual behavior, and fertility potential of rams. We found that biweekly maternal T propionate (100 mg) treatment administered from Day 30-58 of gestation significantly decreased (P < 0.05) postpubertal scrotal circumference and sperm concentration. Prenatal T exposure did not alter ejaculate volume, sperm motility and morphology or testis morphology. There was, however, a trend for more T-exposed rams than controls to be classified as unsatisfactory potential breeders during breeding soundness examinations. Postnatal serum T concentrations were not affected by prenatal T exposure, nor was the expression of key testicular genes essential for spermatogenesis and steroidogenesis. Basal serum LH did not differ between treatment groups, nor did pituitary responsiveness to GnRH. T-exposed rams, like control males, exhibited vigorous libido and were sexually attracted to estrous females. In summary, these results suggest that exposure to exogenous T during the first trimester of gestation can negatively impact spermatogenesis and compromise the reproductive fitness of rams.

Flunixin meglumine improves pregnancy rate in embryo recipient beef cows with an excitable temperament.

Objectives were to determine effects of: 1) handling temperament and administration of flunixin meglumine, an inhibitor of prostaglandin F2a (PGF2a) synthesis, given at the time of embryo transfer, on pregnancy rates in beef cattle embryo transfer recipients; 2) handling temperament and flunixin meglumine on peripheral concentrations of progesterone, cortisol, substance-P, prostaglandin F metabolites (PGFM, (13,14-dihydro-15-keto-PGF2a) and isoprostane 8-epi PGF2a; and 3) flunixin meglumine treatment on proportion of non-pregnant recipient cows returning to estrus within an expected interval. Angus cross beef cows (n = 710) at 7 locations were assigned a body condition score (BCS: 1, emaciated; 9, obese) and a temperament score [0, calm, slow chute exit; walk (n = 352), 1, excited, fast chute exit; jump, trot or run (n = 358)] and were synchronized with Select-Synch with a controlled internal drug release (CIDR) protocol. Estrus detection aids were applied at CIDR removal and cows were observed thrice daily for estrus until 72 h. Recipient cows that expressed estrus and had a corpus luteum received a frozen-thawed embryo on Day 7 after estrus. At the time of transfer, recipient cows were randomly allocated to receive 10 mL of flunixin meglumine im, immediately after transfer (n = 365) or served as an untreated control (n = 345). In a subset of cows (n = 80), ovarian ultrasonography was performed on the day of embryo transfer to determine corpus luteum volume and blood samples were collected twice, at the time of embryo transfer and 7 d later. All cows received estrus detection aids again on Day 14 (7 d after embryo transfer) and were observed for estrus twice daily until Day 24. Accounting for treatment (P > 0.1), embryo transfer difficulty score (P < 0.1), temperament by treatment interaction (P < 0.05), recipient cows with calm temperament had a higher pregnancy rate compared to those with an excited temperament [59.4 (209/352) vs 51.7% (185/358)]. The pregnancy rate for excitable cows without flunixin meglumine was lower (46.3% 81/175) compared to excitable cows that did received flunixin meglumine [56.8% (104/183)], and calm cows that did [59.3% (108/182)] or did not [59.4% (104/170)] receive flunixin meglumine. Proportions of non-pregnant recipient cows returning to estrus on Days 18-24 were not different between flunixin meglumine and control groups, 87.6% (134/153) and 84.0% (137/163), respectively (P > 0.1). At the time of embryo transfer and 7 d later, there were moderate to strong correlations among circulating concentrations of progesterone, cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a. Among excitable cows, progesterone concentrations were lower and cortisol, substance-P, PGFM and isoprostane 8-epi PGF2a concentrations were greater for cows in the control group compared to cows that received flunixin meglumine. In conclusion, administration of flunixin meglumine improved pregnancy rates in excitable recipient cows following embryo transfer without affecting the proportion of non-pregnant cows returning to estrus.

Effects of feeding pregnant beef cows selenium-enriched alfalfa hay on selenium status and antibody titers in their newborn calves.

In newborn dairy calves, it has been demonstrated that supranutritional maternal and colostral Se supplementation using Se yeast or sodium selenite, respectively, improves passive transfer of IgG. In beef cattle, agronomic biofortification with Se is a more practical alternative for Se supplementation, whereby the Se concentration of hay is increased through the use of Se-containing fertilizer amendments. It has been previously demonstrated that agronomic Se biofortification is an effective strategy to improve immunity and performance in Se-replete weaned beef calves. The objective of this experiment was to determine the effects of feeding beef cows Se-enriched alfalfa () hay during the last 8 to 12 wk of gestation on passive transfer of antibodies to calves. At 10 wk ± 16 d before calving, 45 cows were assigned to 1 of 3 treatment groups with 3 pens (5 cows/pen) per treatment: Control cows were fed non-Se-fortified alfalfa hay plus a mineral supplement containing 120 mg/kg Se from sodium selenite, Med-Se cows were fed alfalfa hay fertilized with 45.0 g Se/ha as sodium selenate, and High-Se cows were fed alfalfa hay fertilized with 89.9 g Se/ha as sodium selenate; both the Med-Se and the High-Se groups received mineral supplement without added Se. Colostrum and whole blood (WB) were collected from cows at calving, and WB was collected from calves within 2 h of calving and at 12, 24, 36, and 48 h of age. Concentrations of IgG1 and J-5 antibody in cow colostrum and calf serum were quantified using ELISA procedures. Selenium concentrations linearly increased in WB ( < 0.001) and colostrum ( < 0.001) of cows and in WB of newborn calves ( < 0.001) with increasing Se concentration in alfalfa hay. Colostrum concentrations of IgG1 ( = 0.03) were increased in cows fed Se-biofortified alfalfa hay, but J-5 antibody ( = 0.43) concentrations were not. Calf serum IgG1 ( = 0.43) and J-5 antibody ( = 0.44) concentrations during the first 48 h of age were not affected by prior Se treatment of cows. These data suggest that feeding Se-biofortified alfalfa hay promotes the accumulation of Se and antibodies in colostrum but does not affect short-term serum antibody concentrations in calves.

Effects of Long-Term Flutamide Treatment During Development on Sexual Behaviour and Hormone Responsiveness in Rams.

Testosterone exposure during midgestation differentiates neural circuits controlling sex-specific behaviours and patterns of gonadotrophin secretion in male sheep. Testosterone acts through androgen receptors (AR) and/or after aromatisation to oestradiol and binding to oestrogen receptors. The present study assessed the role of AR activation in male sexual differentiation. We compared rams that were exposed to the AR antagonist flutamide (Flu) throughout the critical period (i.e. days 30-90 of gestation) to control rams and ewes that received no prenatal treatments. The external genitalia of all Flu rams were phenotypically female. Testes were positioned s.c. in the inguinal region of the abdomen, exhibited seasonally impaired androgen secretion and were azospermic. Flu rams displayed male-typical precopulatory and mounting behaviours but could not intromit or ejaculate because they lacked a penis. Flu rams exhibited greater mounting behaviour than control rams and, similar to controls, showed sexual partner preferences for oestrous ewes. Neither control, nor Flu rams responded to oestradiol treatments with displays of female-typical receptive behaviour or LH surge responses, whereas all control ewes responded as expected. The ovine sexually dimorphic nucleus in Flu rams was intermediate in volume between control rams and ewes and significantly different from both. These results indicate that prenatal anti-androgen exposure is not able to block male sexual differentiation in sheep and suggest that compensatory mechanisms intervene to maintain sufficient androgen stimulation during development.

Rumen Microorganisms Decrease Bioavailability of Inorganic Selenium Supplements.

Despite the availability of selenium (Se)-enriched trace mineral supplements, we have observed low Se status in cattle and sheep offered traditional inorganic Se supplements. Reasons for this may include inadequate intake or low bioavailability of inorganic Se sources. The objective of this study was to determine whether rumen microorganisms (RMO) alter the bioavailability of Se sources commonly used in Se supplements. Rumen microorganisms were isolated from ewes (n = 4) and incubated ex vivo with no Se (control), with inorganic Na selenite or Na selenate, or with organic selenomethionine (SeMet). Total Se incorporated into RMO and the amount of elemental Se formed were determined under equivalent conditions. Incorporation of Se from Na selenite, Na selenate, or SeMet into RMO was measured as fold change compared with control (no added Se). Incorporation of Se into microbial mass was greater for SeMet (13.2-fold greater than no-Se control) compared with inorganic Se supplements (P = 0.02); no differences were observed between inorganic Na selenate (3.3-fold greater than no-Se control) and Na selenite (3.5-fold greater than no-Se control; P = 0.97). Formation of non-bioavailable, elemental Se was less for RMO incubated with SeMet compared with inorganic Se sources (P = 0.01); no differences were observed between Na selenate and Na selenite (P = 0.09). The clinical importance of these results is that the oral bioavailability of organic SeMet should be greater compared with inorganic Se sources because of greater RMO incorporation of Se and decreased formation of elemental Se by RMO.

Sex differences in expression of oestrogen receptor α but not androgen receptor mRNAs in the foetal lamb brain.

Gonadal steroid hormones play important roles during critical periods of development to organise brain structures that control sexually dimorphic neuroendocrine responses and behaviours. Specific receptors for androgens and oestrogens must be expressed at appropriate times during development to mediate these processes. The present study was performed to test for sex differences in the relative expression of oestrogen receptor (ER)α and androgen receptor (AR) mRNA during the window of time in gestation that is critical for behavioural masculinisation and differentiation of the ovine sexually dimorphic nucleus (oSDN) in the sheep. In addition, we examined whether ERα and AR mRNA expression is localised within the nascent oSDN and could be involved in its development. Using the quantitative real-time polymerase chain reaction, we found that females expressed more ERα mRNA than males in medial preoptic area and medial basal hypothalamus during the mid-gestational critical period for brain sexual differentiation. No sex differences were found for AR mRNA in any tissue examined or for ERα in amygdala and frontal cortex. Using radioactive in situ hybridisation, we found that the distributions of ERα and AR mRNA overlapped with aromatase mRNA, which delineates the boundaries of the developing oSDN and identifies this nucleus as a target for both androgens and oestrogens. These data demonstrate that the transcriptional machinery for synthesising gonadal steroid receptors is functional in the foetal lamb brain during the critical period for sexual differentiation and suggest that possible mechanisms for establishing dimorphisms controlled by gonadal steroids may exist at the level of steroid hormone receptor expression.

Flaxseed supplementation decreases methanogenic gene abundance in the rumen of dairy cows.

The objective of this study was to investigate the effects of a flaxseed-supplemented diet on archaeal abundance and gene expression of methanogens in the rumen of dairy cows. In all, 11 non-lactating dairy cows were randomly divided into two groups: group A (five cows) and B (six cows). The two diets fed were: (1) the control diet, a conventional dry cow ration; and (2) the flaxseed-supplemented diet, the conventional dry cow ration adjusted with 12.16% ground flaxseed incorporated into the total mixed ration. A cross-over experiment was performed with the two groups of cows fed the two different diets for five 21-day periods, which included the first adaptation period followed by two treatment and two wash out periods. At the end of each feeding period, rumen fluid samples were collected via rumenocentesis and DNA was extracted. Quantitative PCR was utilized to analyze the gene abundance of 16S ribosomal RNA (16S rRNA) targeting the ruminal archaea population and the mcrA gene coding for methyl coenzyme-M reductase subunit A, a terminal enzyme in the methanogenesis pathway. Results demonstrated a 49% reduction of 16S rRNA and 50% reduction of mcrA gene abundances in the rumen of dairy cows fed the flaxseed-supplemented diet in comparison with those fed the control diet. This shows flaxseed supplementation effectively decreases the methanogenic population in the rumen. Future studies will focus on the mechanisms for such reduction in the rumen of dairy cattle, as well as the relationship between methanogenic gene expression and methane production.

The volume of the ovine sexually dimorphic nucleus of the preoptic area is independent of adult testosterone concentrations.

The ovine sexually dimorphic nucleus (oSDN) is characterized by high levels of aromatase mRNA expression which can be used to delineate its boundaries. The volume of the oSDN is approximately 2 to 3-fold larger in rams that mate with ewes (female-oriented rams) than in rams that mate with other rams (male-oriented rams) and ewes. The sex difference in oSDN volume is present in late gestation fetuses and can be eliminated before birth by exposing genetic females to exogenous testosterone during midgestation, suggesting that early exposure to androgen masculinizes volume of the oSDN. The present study was performed to determine whether differences in oSDN volume are influenced by the adult hormonal environment. Adult rams, behaviorally characterized as female-oriented or male-oriented, and ewes were gonadectomized and treated with subcutaneous implants of testosterone to achieve physiologic concentrations of serum testosterone. Three weeks after implant placement brain tissue was prepared for histological assessment of oSDN volume using in situ hybridization for detection of aromatase mRNA expression. Quantitative analysis revealed that despite similar serum testosterone levels among the groups, the volume of the oSDN was greater in female-oriented rams than in male-oriented rams and ewes (P<0.05). Differences in oSDN volume were specific and not reflective of differences in preoptic area height or brain size. These results suggest that differences in the size of the oSDN in adult sheep were not influenced by adult exposure to testosterone.

Changes in LH secretion in response to an estradiol challenge in male- and female-oriented rams and in ewes.

Two experiments were conducted to determine whether an estradiol challenge could cause a female-type LH surge in castrated male- and female-oriented rams (MORs and FORs). Administration of 17beta-estradiol to castrated MORs and FORs and ovariectomized ewes caused an initial reduction in LH secretion followed for 12-20 h by a surge release of LH in the ewes. No surge release of LH occurred in the MORs and FORs. The pattern of changes in LH secretion within rams and ewes did not differ between the breeding and nonbreeding seasons. Treatment failed to elicit female-typical receptive sexual behaviors in the rams but did stimulate increased sexual receptivity in the ewes as determined by the measures of responsiveness to the teaser ram. Overall, no differences were found in hypothalamic-hypophyseal function in response to exogenous estradiol between MORs and FORs. These data are interpreted to suggest that in contrast to sexual attraction, the neural mechanisms controlling the LH surge and female receptivity are defeminized in MORs.

Does increased PGF2 alpha receptor concentration mediate PGF2 alpha-induced luteolysis during early diestrus in the pig?

The mechanism by which multiple injections of PGF2 alpha result in premature luteolysis in pigs is unknown. In the present study we evaluated whether PGF2 alpha receptor concentrations on large luteal cells changed when gilts were injected IM with 12.5 mg of PGF2 alpha every 12 hours from the morning of day 5 of an estrous cycle (estrus = day 0) until ovariectomy on day 6, 7, 8, or 9. Luteal PGF2 alpha receptor concentrations remained constant from day 6 through 9 in the PGF2 alpha-treated group, but increased linearly (P > 0.05) in control gilts from day 6 to 9. Receptor affinity for PGF2 alpha did not change throughout the study in either PGF2 alpha-treated or control gilts. Luteal progesterone concentrations were significantly lower in PGF2 alpha-treated gilts than in control gilts only on day 9. Histological examination of luteal tissue obtained from PGF2 alpha-treated gilts revealed definite evidence of luteolysis by day 8. We conclude that PGF2 alpha-induced premature luteolysis is not mediated by an increase in luteal PGF2 alpha receptor concentrations and, based on luteal progesterone concentrations and histological features, that the PGF2 alpha-based protocol used to shorten the estrous cycle is accompanied by premature functional and structural luteal regression.

Infertility due to abnormalities of the ovaries in cattle.

A careful physical examination of a cow or heifer suspected of having an ovarian problem often results in a specific diagnosis (e.g., freemartinism) or a workable list of differentials. When the diagnosis is uncertain, techniques such as rapid progesterone assays, ultrasonographic imaging, ova or embryo recovery, and cytogenetic evaluation can provide critical information for a well-based diagnosis and prognosis. Despite the wide array of problems that can afflict the bovine ovary, cystic ovaries probably are the most commonly diagnosed and treated ovarian abnormality. Cysts have a variable life span and sometimes occurs together with a CL. Hormonal therapy with either GnRH followed by PG approximately 9 to 14 days later, or GnRH alone, followed by good heat detection is the treatment of choice for cows with cysts. Other conditions associated with ovarian dyfunction and infertility include adhesions, developmental anomalies, and tumors. The life span of a CL in the cow can be shortened unintentionally by attempting to correct other problems (e.g., multiple injections of oxytocin for milk let-down), or lengthened by uterine pathology (e.g., pyometra, or uterus unicornis). The administration of GnRH or hCG to improve ovarian function and pregnancy rates in cows may be beneficial in selected herds.

Repeated administration of prostaglandin F2 alpha during the early luteal phase causes premature luteolysis in the pig.

Previous investigators considered pig corpora lutea refractory to the luteolytic effects of prostaglandin (PG) F2 alpha before Day 12 of the estrous cycle. This study was designed to determine whether multiple injections of PGF2 alpha would result in a sustained reduction of serum progesterone and luteolysis, leading to significant shortening of the estrous cycle and interestrous interval. On Days 5-10 of an estrous cycle, gilts (n = 4) received injections of 12.5 mg PGF2 alpha (dinoprost tromethamine) i.m. every 12 h, or vehicle (PBS; n = 4) according to the same schedule. Mean interestrous interval in PGF2 alpha-treated gilts was reduced (p < 0.001) to 13.3 +/- 0.5 days compared with 19.8 +/- 0.6 days for control gilts. Serum progesterone declined below 1 ng/ml by Day 10.5 in PGF2 alpha-treated gilts compared to Day 17.5 in control animals. Serum concentrations of estradiol-17 beta (E2) reached maximal levels in PGF2 alpha-treated gilts earlier (Day 12.5) in the cycle than in control gilts (Day 19.5). Peak E2 and LH concentrations coincided with the periestrous period, suggesting that PGF2 alpha-induced estrus is accompanied by normal follicular development and ovulation. These results demonstrate that the pig is susceptible to the luteolytic effects of PGF2 alpha before Day 12 if repeated injections are given from Day 5 through Day 10.

Transendoscopic Nd:YAG laser ablation of vaginal septa in a bitch.

A vaginal septum was diagnosed in an English bulldog bitch during routine estrous cycle staging. The septum extended 16 cm from the vestibulo-vaginal junction to just caudal to the cervix. The septum was removed by two applications of an Nd:YAG laser via a flexible fiberoptic endoscope. Complete healing occurred over a 5-week period and was uneventful. The bitch was bred and subsequently delivered four puppies vaginally. Transendoscopic laser ablation provided a noninvasive approach to surgically remove a vaginal septum in the dog.