Control Rams Research Articles

Birth weight, growth indices, and seminal parameters in male offspring are resilient features to maternal pre-conceptional dietary manipulation in sheep

Gestational diet manipulation can lead to inadequate fetal nutrient supply resulting in low birth weight, limited postnatal growth, and consequently, reduced reproductive performance in the progeny. However, effects of short-term maternal pre-conceptional dietary manipulation on postnatal growth and reproductive parameters of male offspring in large animals remains unexplored. To determine these consequences, female crossbred (Polypay x Dorset) sheep were allocated to three groups (n = 33/group) of dietary manipulation for 21 days prior to mating under the following conditions: (1) control at 100 % of maintenance energy requirements (40 Kcal of metabolizable energy/kg body weight [BW]), (2) undernutrition (UN) at 50 % of Control intake, and (3) overnutrition (ON) at 200 % of maintenance energy. Singleton ram lambs (UN:9; C:12; ON:6) were monitored from birth until 8 months of age, including birth weight, weekly weights, weight gain, body mass index (BMI), and circulating testosterone. After weaning, monthly scrotal circumference and subcutaneous fat depth were measured. Semen morphology and motility were evaluated at 7 and 8 months of age. Birth weight, weight gain, and BMI at birth and weaning were not significantly different among nutritional treatments. None of the pre-conceptional diets affected body weight change from weaning until 36 weeks of age, BMI, fat depth, or scrotal circumference across the experiment. A sustained rise in plasma testosterone concentrations was detected when ram lambs were, on average, 82 days old and 37 kg. Both testosterone concentrations and scrotal circumference were positively correlated to body weight regardless of treatment group. In addition, seminal parameters did not differ among treatments, but a transient increase in plasma testosterone at 18 weeks of age was observed in ON ram lambs compared to control rams. In conclusion, birth weight, growth indices, and seminal parameters in singleton rams are resilient features in the progeny upon maternal pre-conceptional dietary manipulation in sheep.

A single administration of carbetocin before electroejaculation increases the insemination doses produced from each ejaculate in rams

The aim of the study was to determine the effect of carbetocin administration (a long-acting analog of oxytocin) 20 or 10 min before electroejaculation (EE) on the duration of semen collection procedure, quantitative and qualitative characteristics of the ejaculate, and stress biomarkers in rams. Semen was collected from 12 Corriedale rams (age, 2.5–5.5 years old) with EE, in a Latin-square design, administrating carbetocin (0.2 mg/100 kg of body weight i.v.) 20 or 10 min before EE, or without carbetocin administration (CB-20, CB-10, and CON treatments, respectively). Each treatment was applied to different rams every 3–4 days, allowing all the rams to receive all three treatments. Carbetocin administered 20 or 10 min before EE increased the number of sperm ejaculated (P = 0.01), the semen concentration (P = 0.02), the number of insemination doses collected in a single collection (P = 0.01), and the number of insemination doses collected/electrical pulses administered (P = 0.05) compared to control rams. Carbetocin administered 20 or 10 min before semen collection prolonged the time required for EE and the number of pulses administered during EE compared to CON rams (P < 0.03 for both). The CB-10 rams required the administration of more electrical pulses during ejaculation than CON rams (P = 0.001), and CB-20 treatment tended to require more electrical pulses than CON rams (P = 0.06). The volume of the ejaculate was greater in CB-10 than in CON rams (P = 0.01), and that of CB-20 treatment tended to be greater than CON rams (P = 0.08). The percentage of sperm with intact membrane was greater in CB-20 than in CON rams (P = 0.01). Total protein, albumin, and globulin concentrations were lower immediately after carbetocin administration 20 or 10 min before EE. The treatments did not affect cortisol concentration, glycemia, rectal and surface temperatures, heart rate, and facial expressions. Carbetocin administration before EE of rams improved the quantitative and qualitative characteristics of the ejaculate, duplicating the number of insemination doses collected. It can be a promising treatment to obtain a greater quantity of doses to inseminate with a lower frequency of semen collections, reducing the negative impacts of EE on animal welfare.

Semen quality of rams fed a liposomal vitamin-mineral supplement during the period of sexual rest

To increase the sexual activity and sperm quality of rams during the period of sexual rest, it is necessary to increase the consumption rates of vitamins and microelements to the level of the mating season. The aim of the work was to find out the effect of feeding a liposomal vitamin-mineral supplement on the quality parameters of ram sperm during the period of sexual rest. The experiment was conducted on 12 clinically healthy Texel rams, aged 2–4 years, during the period of sexual rest (March–May). Animals were divided into two groups: control and experimental, 6 heads each. The control rams received the basic diet, the animals of the experimental group were individually added to the combined feed for 45 days with a feed additive in the form of a liposomal emulsion, which included: vitamins A, D3, E, C and zinc gluconate. After the end of supplement feeding, ejaculates were collected from the rams for research. The motility, kinetic indicators of sperm, the activity of succinate dehydrogenase (SDH) and cytochrome oxidase (CO) in sperm, as well as the activity of antioxidant protection enzymes superoxide dismutase (SOD), glutathione peroxidase (GPO) and catalase (CAT) were determined. It was established that feeding liposomal vitamin-mineral supplement to rams during the period of sexual rest significantly (P &lt; 0.05) increases sperm activity, their kinetic indicators: curvilinear (VCL), straight-line (VSL) and average speed (VAP) of sperm movement (P &lt; 0.05–0.001), the activity of enzymes – markers of the fertilizing capacity of SDH and CO sperm (P &lt; 0.05–0.001). At the same time, there was a significant (P &lt; 0.05) decrease in the activity of SOD with a simultaneous significant (by 23.3–25.0 %) increase in the activity of GPO and CAT (P &lt; 0.01). Higher indicators of the quality of ejaculates of rams under the influence of vitamins A, D3, E, C and zinc gluconate indicate the possibility of obtaining high-quality sperm from breeding rams during the period of sexual rest.

Incorporation of fresh leaves of wormwood (Artemisia herba alba) and/or rosemary (Rosmarinus officinalis) in the diet of rams: Effect on testicular function, sexual behavior, and blood parameters.

This study aimed to investigate the effect of wormwood and rosemary supplementation on some reproductive traits of Barbarine rams. The experiment lasted 2 months. Twenty-four adult rams were divided into four groups (n=6) balanced for the weight (53.3 ± 1.2 kg body weight [BW] ± SD). All rams received 1200 g of straw and 600 g of barley. Control rams (C) without aromatic medicinal plant (AMP), while experimental rams received 20 g of fresh rosemary leaves (R), 20 g of fresh wormwood leaves (A), and 10 g of fresh rosemary leaves plus 10 g of fresh wormwood leaves (RA). The results revealed that the live weight of all rams increased (p < .05) in the RA group compared to the C, A, and R groups. Scrotal circumference increased in the R rams when compared to the controls rams (p < .05). For sperm parameters we showed that the A rams had higher sperm concentrations (p < .05). But, the sperm volume decreased in the R rams (p > .05). However, when the rams received rosemary plus wormwood, their sperm volume increased (p > .05). The sperm mass motility was higher for the A, R and AR rams in comparison to the C rams (p=.05). On the other hand, biochemical analysis of the seminal fluid showed no effect of diets on calcium and total proteins concentration. But the measurement of glucose and seminal insulin showed a decrease (p < .05) in these two biochemical markers in group A rams and a decrease (p < .05) in insulin without modification of the glucose concentration in R rams. Blood glucose and insulin decreased in the animals on AMP diet compared to the other groups (p < .05) while aspartate aminotransferase (AST) increased (p < .05). Rosemary leaves (R and RA groups) increased (p < .05) plasma cortisol compared to the other groups. It can be concluded that the addition of Rosmarinus officinalis and/or Artemisia herba alba in ram diet can have a positive effect on the reproductive function by increasing the concentration and motility of sperm, plasma testosterone, and sexual behavior.

Low-moderate dietary phytoestrogens transiently disrupt spermatogenesis and the seminal plasma proteome in the ram.

Dietary phytoestrogens disrupt a specific stage of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa. This paper demonstrates for the first time that ram reproduction is compromised by oestrogenic pasture, whilst also providing a longitudinal model for the impact of phytoestrogens on male fertility. Compounds with oestrogen-like actions are now common in both the Western diet. The long-term impacts and underlying mechanisms by which oestrogenic compounds alter male reproduction, however, are unclear. To investigate this, we used a longitudinal sheep model examining the impact of oestrogenic pasture consumption on semen quality and production, testicular size, sexual behaviour and the seminal plasma proteome of Merino rams (n = 20), over a full spermatogenic cycle and in the subsequent breeding season. Throughout the study period, sexual behaviour, sperm production and motility were similar between the exposed and non-exposed rams (P > 0.05). However, between 5 and 8 weeks of exposure to dietary phytoestrogens, rams produced a higher percentage of spermatozoa with a specific malformation of the sperm midpiece and reduced DNA integrity, compared to non-exposed rams (P < 0.001). Investigation into the seminal plasma proteome revealed 93 differentially expressed proteins between phytoestrogen-exposed and control rams (P < 0.05). Exposure to phytoestrogens increased the expression of proteins involved in cellular structure development, actin cytoskeleton reorganisation, regulation of cell function and decreased expression in those related to catabolic processes. The greatest fold changes were in proteins involved in the assembly of the sperm flagella, removal of cytoplasm, spermatid development and maintenance of DNA integrity. After returning to non-oestrogenic pasture, no differences in any measure were observed between treatment groups during the subsequent breeding season. We conclude that dietary phytoestrogens can transiently disrupt specific stages of ram spermatogenesis, causing subtle decreases in sperm quality by affecting the expression of pathways involved in the structural integrity of the spermatozoa.

Effects of paternal methionine supplementation on sperm DNA methylation and embryo transcriptome in sheep.

Environmental effects on gene expression and offspring development can be mediated by epigenetic modifications. It is well established that maternal diet influences DNA methylation patterns and phenotypes in the offspring; however, the epigenetic effects of paternal diet on developing offspring warrants further investigation. Here, we examined how a prepubertal methionine-enriched paternal diet affected sperm DNA methylation and its subsequent effects on embryo gene expression. Three treatment and three control rams were bred to seven ewes, and blastocysts were flushed for RNA extraction. Semen was collected from all rams and submitted for reduced representation bisulfite sequencing analysis. In total, 166 differentially methylated cytosines were identified in the sperm from treatment versus control rams. Nine genes were found to be differentially expressed in embryos produced from treatment versus control rams, and seven differentially methylated cytosines in the sperm were found to be highly correlated with gene expression in the embryos. Our results demonstrate that sperm methylation differences induced by diet may influence fetal programming.

Rams sexually activated in spring by light treatment stimulate social and sexual activities in non-activated rams

Twelve rams were used to confirm whether rams sexually activated in spring by light treatment increase social and sexual activities of non-treated rams in the presence of ewes. Males were divided into 2 groups: light-treated rams (group L, n = 6), which were exposed to artificial long days (16 h light) for 2 months (1 Nov-31 Dec), or non-exposed, control rams (group C, n = 6), exposed to the natural photoperiod. At the end of the long-day period, light-treated rams were returned to the natural photoperiod, groups isolated from each other. In mid-Feb, 3 rams from each group were put together such that 4 experimental groups were created: the remaining light-treated rams (L, n = 3), the remaining control rams (C, n = 3), and the other treated (L-mixed n = 3) and non-treated (C-mixed, n = 3) rams, exposed to 30, 30, and 60 ewes, respectively, in 3 separate paddocks. To identify each ram, large (30-cm-high) numbers were painted on the sides and rump of the males with washable sheep spray paint. Behaviors were documented by 4 video cameras using 6 30-minute segments from 1600 to 1800 h the following day (27 hours). C rams presented the lowest proportion of activities (P < 0.01), and C-mixed rams exhibited proportions similar to L and L-mixed rams. C rams exhibited the fewest fight attempts (P < 0.001), fighting (P < 0.05), pushing (P < 0.001), and sniffing another ram. The groups differed significantly (P < 0.001) in pushing, pawing, flehmen, and attempt mounting, and C rams had the fewest interactions. In conclusion, rams exposed to 2 months of long days at late-autumn early-winter increased social and sexual activities of unexposed rams. These observations can be explained by a “ram-to-ram effect”, which can enhance the endocrine activity of rams in sexual rest if they are joined with rams who have been made sexually active by a photoperiodic treatment. These findings suggest that socio-sexual interactions could be used to restrict out-of-season reproduction in this species.

Гематологічні показники та якість сперми баранів у період статевого спокою за згодовування ліпосомальної вітамінно-мінеральної добавки

The aim of the work was to investigate the effect of feeding a liposomal vitamin and mineral supplement during the period of sexual rest on hematological indicators and quality of ram sperm. The experiment was conducted at the PE “Kogut BM” in Horodotsky district, Lviv region, on 12 clinically healthy Texel rams, aged 2–4 years, during the period of sexual rest (March-May). Animals were divided into two groups — control and experimental, 6 animals per group. The control rams received the basic diet containing hay, corn silage, and compound feed. For 45 days, the sheep of the experimental group were individually added to the combined feed a feed additive containing vitamins A, D3, E, C and zinc gluconate in the form of a liposomal emulsion. At the beginning and at the end of feeding, we took blood samples, in which hematological indicators were determined. After the end of supplement feeding, ejaculates were collected twice a week in doublets for three weeks. Physiological indicators of ejaculate quality (volume, sperm concentration, percentage of live sperm, as well as sperm viability, morphological disorders and percentage of degenerative sperm) were determined using the CASA computerized system. It was established that feeding liposomal vitamin-mineral supplements to rams during the period of sexual rest improves hematological indicators: the content of erythrocytes, hemoglobin, platelets and hematocrit increase significantly (P&lt;0.05–0.001) while the content of leukocytes decreases by 24.2% (P&lt;0.05). Accordingly, the erythrocyte indices of the blood of experimental breeding rams were higher than those of control animals. Feeding rams with a liposomal vitamin and mineral supplement ensured an increase in ejaculate volume by 17.6% (P&lt;0.05), sperm concentration (P&lt;0.01), their viability, as well as a decrease in the number of immature (P&lt;0.001) and degenerated (P&lt;0.05) sperm. Higher quality indicators of ejaculates of rams under the influence of vitamins A, D3, E, C and zinc gluconate indicate the possibility of obtaining sperm from breeding rams during the period of sexual rest.

Intramuscular administration of l-arginine boosts testicular hemodynamics, plasma concentrations of testosterone and nitric oxide in heat-stressed rams

The current study aimed to assess, for the first time, the effects of intramuscular injection of l-arginine (L-arg) on testicular hemodynamics, echogenicity, and plasma concentrations of testosterone, total antioxidant capacity, and nitric oxide (NO) in Ossimi rams. Twelve sexually matured heat-stressed rams were randomly assigned to one of two groups: the L-arg group (n = 6) received 5 mg/kg L-arg dissolved in 2 ml normal saline 0.9%, or the control group (n = 6) received merely 2 ml of normal saline 0.9%. Blood sampling, B-mode ultrasound assessment of the testicular parenchyma, and pulsed-wave Doppler ultrasound of the testicular artery for both right and left testis were performed immediately before 0 min and 1, 4, 24, 48, 72, and 168 h after L-arg or saline administration. In the L-arg group, resistive index (RI) and pulsatility index (PI) means were significantly lower compared to the control group at 4–168 h post-treatment. Plasma testosterone concentrations were higher (P < 0.05) at 4 h and onward in the L-arg treated compared to the control rams, the same for NO levels however its increase (P < 0.05) was observed as soon as 1 h post-treatment. In L-arg treated rams, NO concentrations were positively correlated to plasma testosterone concentrations (r = 0.7, p < .01), but negatively correlated to both RI and PI (r = −0.8 and −0.6, respectively, p < .01). In conclusion, l-arginine administration enhanced testicular blood flow and increased plasma testosterone and nitric oxide concentrations in heat-stressed rams.

Sexual Preference of Crossbreed Ewes for Rams Medicated with Naloxone

Ewe sexual preference depends on several factors; ram attractiveness is important. This work was carried out in in the first week of September 2020 and repeated in November 2021. For the first trial two rams were treated with 0.5 mg naloxone im for 15 days, two control rams were treated with saline injections. Sexual preference was studied with synchronized ewes (n 0 12) displaying estrus at the end of naloxone treatment of the rams. Ewe’s proceptive and receptive behavior were noted. Plasma testosterone levels were monitored through the duration of the experiment in treated and control rams. For the second trial 4 rams and ewes were treated same as trial 1, and the number of ewes was increased to 24. It was observed that on the first week of treatment, testosterone levels were not significantly different between treated and control rams; but at the beginning of the second week of treatment testosterone plasma levels in naloxone treated rams increased until values reached levels of 15 ng/mL compared with 6 ng/mL in control rams. When ewes were exposed to treated and untreated rams during the first trial, 11 ewes showed high proceptivity and receptivity for the naloxone treated ram (p&lt;0.01) and they were mated, for the control ram only one ewe showed complete preference. In the second trial it was observed that 19 ewes also showed high proceptivity and receptivity for naloxone treated rams (p &lt; .001). Hormone levels behaved following the same pattern as that observed during the first trial. It was concluded that naloxone treatment facilitated the secretion of testosterone in rams and the high concentration of this androgen motivated ewes to prefer mating with the naloxone treated ram.

Basic Exploratory Study of Bisphenol A (BPA) Dietary Administration to Istrian Pramenka Rams and Male Toxicity Investigation.

Bisphenol A (BPA), an endocrine-disrupting chemical and environmental pollutant, has been reported by many researchers to induce male reproductive toxicity in different experimental models. In this study, we investigated whether long-term exposure for two months to 25 µg/kg body weight (low dose) of BPA affects spermatogenesis or sperm quality in young Istrian Pramenka rams exposed via diet. We evaluated body and testicular weights, histopathology of testes and epididymides, and sperm analyses, and compared these parameters between the group of treated rams and the control group of rams. Although there were some differences between the two groups, these differences were not large or statistically significant. The only statistically significant difference was the lower epithelial height of seminiferous tubules in treated rams, compared to control rams. In addition to assessing toxicity, BPA concentrations in the blood plasma of treated rams were determined after the first administration, and the toxicokinetic parameters of total BPA were calculated. In this study, no major signs of altered reproduction in rams were detected.

Infection of West African dwarf rams with Trypanosoma brucei brucei and Trypanosoma congolense significantly alter serum electrolytes, redox balance, sperm parameters, and gonadal morphology.

Trypanotolerance of the West African dwarf (WAD) breeds may not rule out significant pathophysiological changes that may affect productivity. In this study, the effects of infection of WAD rams with Trypanosoma brucei brucei (Tbb) and Trypanosoma congolense (Tc) on their serum levels of electrolytes [calcium, phosphorus, sodium, potassium]; oxidative stress markers [superoxide dismutase (SOD), malondialdehyde (MDA)]; and sperm parameters [sperm count, motility, vitality, and morphology] were investigated. Fifteen WAD rams, assigned to 3 groups (A, B & C) of 5 rams each, were used for the study. Group A rams were infected with Tbb, while Group B rams were infected with Tc, both intraperitoneally, at the dose of 106 trypanosomes/animal. Group C rams served as the uninfected control. The infections were monitored for 70days. Serum calcium levels were significantly (p < 0.05) lower in Tbb and Tc infected rams compared to the control throughout the study. Serum sodium was significantly (p < 0.05) higher in the Tb infected rams compared to the Tc infected and control rams on days 14 and 28 PI. Serum SOD activity decreased while MDA levels increased in both infected groups of rams. Tbb infected rams were azoospermic, while Tc infected rams had lower sperm motility, vitality and concentration, and higher number of abnormal sperm cells compared to the control. Necrotic and inflammatory lesions occurred in the testis and epididymis of both infected rams. These results suggest that despite trypanotolerance, trypanosome infections in the WAD rams significantly impact on health and reproduction.

Effects of melatonin administration on testicular hemodynamics, echotexture, steroids production, and semen parameters during the non-breeding season in Ossimi rams

This study was conducted to determine the influence of a single subcutaneous (SC) injection of melatonin (18 and 36 mg) on testicular hemodynamics, echotexture, plasma testosterone concentration, and sperm quality characteristics during the non-breeding season in Ossimi rams. Eighteen sexually mature, Ossimi rams were randomly allocated into two treated groups that received either 18 mg (Mel 18 group; n = 6) or 36 mg (Mel 36 group; n = 6) of melatonin powder dissolved in 1 ml corn oil (SC) and a control group (n = 6) that received 1 ml corn oil only. Blood collection and ultrasonographic assessment of the testes and supratesticular arteries were conducted immediately before treatment (W0) and once weekly for 8 successive weeks after melatonin injection (W1–W8). Resistive index (RI) and pulsatility index (PI) values were markedly decreased (P ˂ 0.05) within Mel 36 (at W4–W8) and e Mel 18 (at W8) groups, which was not observed within the control group (P ˃ 0.05). Testicular echogenicity was severely reduced (P ˂ 0.05) in Mel 36 (at W4–W5) and Mel 18 (at W8) groups, which was not occurred in the control one (P ˃ 0.05). The concentration of plasma testosterone was increased (P ˂ 0.05) in Mel 36 (at W3–W8) and Mel 18 (at W8) groups, which was not clear for the control rams (P ˃ 0.05). The percentage of progressively motile sperm cells was higher (P ˂ 0.05) in Mel 36 group (at W5–W8), which was not recorded within the Mel 18 or control groups (P ˃ 0.05).The percentages of sperm cells viability and normal morphology were higher (P ˂ 0.05) within Mel 36 (at W6–W8) and Mel 18 (at W7–W8) groups, which was not detected within the control group (P ˃ 0.05).In conclusion, the administration of melatonin ameliorates the testicular function via enhancing the testicular vascular irrigation, testosterone production, and sperm cells’ quality parameters in Ossimi rams. So, we recommend administration of a single SC dose of melatonin (36 mg) at least one month before using rams for breeding especially out of the reproductive season.

Research of the overall chemical and amino acid composition of meat from young animals of new sheep genotypes

The paper presents the results of the investigation of the overall chemical composition (mass fraction of moisture, fat, protein and ash) and the amino acid composition of mutton from the experimental animals of new genotypes: Sovetsky Merino x Dzhalginsky Merino and Sovetsky Merino x Stavropolsky breed. As a control, meat of purebred Sovetsky Merino was studied. The experimental and control rams were slaughtered at the age of 8 months by the conventional technology. Meat samples for laboratory examination were taken a day after slaughter. The results of the analysis of the overall chemical composition showed that lamb meat of new genotypes had lower moisture content and higher content of fat and protein compared to the control. Analysis of the amino acid composition of meat proteins of genotypes Sovetsky Merino x Dzhalginsky Merino and Sovetsky Merino x Stavropolsky breed revealed lower content of essential amino acids compared to the control. Comparison of amino acid scores showed that the amino acid composition of meat proteins from the experimental animals was limited by valine and lysine, respectively. It was concluded by the results of the investigations of the amino acid composition of proteins that the biological full value of proteins of mutton from new genotypes was 22.9% and 30.3% lower than that of the control.

Daily variation of the cortisol and insulin concentration, glycemia, surface temperature, and behavior patterns due the administration of equine Chorionic Gonadotropin in rams

Sex steroids can modulate the circadian rhythmicity of some physiological and behavioral functions of sheep. During the non-breeding season, Corriedale rams have a low testosterone concentration, but the administration of equine Chorionic Gonadotropin (eCG) stimulates its secretion. Therefore, the daily variation of cortisol and insulin concentrations, glycemia, behavioral pattern (eating, ruminating, standing, lying, and sleeping), and rostral and eye surface temperatures were compared in rams treated or not treated with eCG during the non-breeding season [between August (late winter) and September (early spring)]. Twenty Corriedale adult rams (BW 63.4 ± 7.3 kg; BCS: 3.5 ± 0.5; mean ± SD; age: 2-3 years old) were randomly assigned to 1 of 2 treatments: a) Treated: administration of 1000 IU of eCG intramuscularly and b) Control: control, without application of eCG. All the rams were managed under the same feeding and environmental conditions (individual pens outdoors). All variables were measured each hour for 26 h, except testosterone and insulin that were measured at 09:00, 10:00, 12:00, 15:00, 18:00, 21:00, 22:00, 24:00, 03:00, 06:00, 09:00 and 11:00 h. Testosterone and cortisol concentration were greater in Treated than Control rams (testosterone: 32.7 ± 0.7 nmol/L vs 5.8 ± 0.7 nmol/L; P <0.0001; cortisol: 11.3 ± 0.6 nmol/L vs 9.9 ± 0.6 nmol/L; P =0.02). The eye surface temperature was 0.9°C lower in the Treated than in the Control rams (P =0.004). The characteristics of the daily variation pattern of cortisol, glycemia, glycemia:insulin ratio, and surface temperature were not affected by the treatment, the acrophases being at ∼06:00 h, ∼18:00 h, ∼08:00 h, and ∼11:00 h, respectively. The acrophase of insulin concentration was observed earlier in Treated than in Control rams (13:02 ± 0:56 vs 15:47 ± 0:53; P =0.04). Additionally, the acrophase of the time spent eating was advanced in Treated rams (09:48 ± 00:16 vs 10:20 ± 00:16; P =0.04) and sleeping (21:39 ± 00:57 vs 02:02 ± 01:06; P <0.0001). Treated rams slept less time than Control rams (P =0.04). In conclusion, the administration of eCG modulated the daily variation pattern of insulin concentration and the behavioral pattern (period in which rams were observed eating and sleeping). Nevertheless, although there was a greater cortisol concentration and reduced eye surface temperature, the administration of eCG did not modify their daily variation pattern, suggesting that this is robust, limiting the effect of testosterone on their basal pattern.

The Intergenerational Impacts of Paternal Diet on DNA Methylation and Offspring Phenotypes in Sheep.

Knowledge of non-genomic inheritance of traits is currently limited. Although it is well established that maternal diet influences offspring inheritance of traits through DNA methylation, studies on the impact of prepubertal paternal diet on DNA methylation are rare. This study aimed to evaluate the impact of prepubertal diet in Polypay rams on complex traits, DNA methylation, and transmission of traits to offspring. A total of 10 littermate pairs of F0 rams were divided so that one ram was fed a control diet, and the other was fed the control diet with supplemental methionine. Diet was associated with earlier age at puberty in treatment vs. control F0 rams. F0 treatment rams tended to show decreased pubertal weight compared to control rams; however, no differences were detected in overall growth. A total of ten F0 rams were bred, and the entire F1 generation was fed a control diet. Diet of F0 rams had a significant association with scrotal circumference (SC) and weight at puberty of F1 offspring. The paternal diet was not significantly associated with F1 ram growth or age at puberty. The DNA methylation of F0 ram sperm was assessed, and genes related to both sexual development (e.g., DAZAP1, CHD7, TAB1, MTMR2, CELSR1, MGAT1) and body weight (e.g., DUOX2, DUOXA2) were prevalent in the data. These results provide novel information about the mechanisms through which the prepubertal paternal diet may alter body weight at puberty and sexual development.

Administration of equine Chorionic Gonadotrophin (eCG) to rams to improve the reproductive performance during the non-breeding season

In most ram breeds, there are annual changes in testosterone serum concentration and semen quality. This study aimed to determine if eCG administration to rams during the non-breeding season improves the reproductive activity. In the first study, we compared the effects of administering three doses of eCG (0, 400 or 700 IU). In the second experiment we compared the effects of administering eCG to rams from two breeds originated in different latitudes, and thus, with different expected reproductive seasonal pattern (Highlander; HL and Texel; TEX). The first study was performed during the mid non-breeding season with three groups of rams: treated with 400 IU (group eCG400) or 700 IU of eCG (group eCG700), or untreated controls (group CON). Treated rams received three doses of eCG administered every 6 days. Testosterone concentration, testicular traits (scrotal circumference, testicular and sexual skin flush, and ultrasonographic testes pixel color intensity) and seminal characteristics were determined from five days before the first eCG administration until Day 24. Testosterone concentration was greater in eCG700 than in eCG400 (P <0.001) and CON (P <0.0001) rams, and in eCG400 than in CON rams (P = 0.0002). However, the treatment did not modify the testicular traits and seminal quality. The second study was performed with 15 HL and 17 TEX rams; while 8 HL and 9 TEX rams received two doses of 1000 IU of eCG (Days 0 and 5), the other rams remained as untreated controls. Scrotal circumferences, seminal traits (fresh and thawed semen, and cryopreservation ratio –CR-) as well as the sexual behavior were determined, twice before eCG administration and during 3 weeks. The treatment did not modify the scrotal circumference nor the fresh sperm traits evaluated. However, the CR of motile and progressive motile sperm were greater in eCG treated rams than in control rams on Day 7 (P = 0.01 for both). Treated rams had their second and third ejaculations earlier than control rams (P = 0.001 and P = 0.02; respectively). In conclusion, the administration of eCG during the non-breeding season stimulated testosterone secretion without modifying the main reproductive outcomes of rams. Reducing the interejaculatory intervals could be considered for intensive semen collection programs.

Effects of TLR4 overexpression on sperm quality, seminal plasma biomarkers, sperm DNA methylation and pregnancy rate in sheep

Genetic modification provides a means to enhancing disease resistance in animals. In this study, the first generation of genetically modified (GM) sheep overexpressing TLR4 was produced by microinjection for better disease resistance. To compare semen characteristics including sperm quality, seminal plasma biochemical index, sperm DNA methylation and pregnancy rate of three-year old transgenic sheep with TLR4 overexpressed (toll like receptor 4, TLR4) and non-transgenic ram. Sixteen transgenic ram of F0 generation were produced by microinjection of the TLR4 plasmid into the pronucleus of fertilized ova. Seven transgenic sheep of F1 generation was produced by breeding F0 transgenic founders with non-transgenic sheep of the same breed. There were no significant differences between transgenic and control rams for all semen quality parameters, including semen volume, sperm concentration, sperm viability, and percentages of sperm with an intact plasma membrane, acrosomal integrity, and viable sperm with high mitochondrial membrane potential in both F0 and F1 generation. Furthermore, no significant differences were found for seminal plasma concentrations of zinc, neutral alpha-glucosidase, acid phosphatase or fructose, nor for levels of H19 and IGF2R methylation in sperm DNA. In addition, pregnancy rate was also similar between these two groups. In conclusion, there was no evidence that TLR4 overexpression altered the sperm quality, seminal plasma or sperm DNA of transgenic sheep.

Light-induced sexually active rams prevent the seasonal inhibition of luteinizing-hormone in ovariectomized estradiol-implanted ewes

The effect of the continuous presence of sexually active Rasa Aragonesa rams on the plasma luteinizing-hormone (LH) concentrations of ewes was studied from November to May. Light-treated rams were rendered sexually active (SA rams) by exposure to 2 months of artificially long days (16 h light/8 h dark) in one of two groups from either 1 November (SAR1, n = 3) or 1 December (SAR2, n = 3). Rams (n = 6) in a Control group were kept under the natural photoperiod. Thirty ewes were ovariectomized in September and implanted with a subcutaneous implant (l = 15 mm) that contained estradiol-17β. One group of ewes (SAR; n = 10) was housed with control rams from 1 October to 15 February before being housed with SAR1 rams from 16 February to 31 March, and with SAR2 rams from 1 April to 31 May. A second group of ewes (C; n = 10) remained with control rams throughout the experiment, and a third group was kept isolated from rams throughout the experiment (ISO; n = 10). Blood samples were collected weekly from November to May, and plasma LH concentrations were measured. In the breeding season (November–February), plasma LH concentrations of ewes did not differ significantly between groups (SAR: 2.00 ± 0.34; C: 1.88 ± 0.16; ISO: 1.67 ± 0.51 ng/ml). From March to May (seasonal anestrus), however, LH plasma concentrations decreased in the C and ISO groups (1.30 ± 0.20 and 0.48 ± 0.04 ng/ml, respectively), but remained at the same level as in the breeding season in the SAR group (2.30 ± 0.17 ng/ml; P < 0.001). Significant differences (P < 0.01) between groups were observed from March onwards: LH concentrations were highest in SAR ewes (P < 0.001) and lowest in the ISO ewes. In conclusion, the continuous presence of sexually active rams prevented the seasonal decrease in plasma LH concentrations, probably by preventing the seasonal negative feedback of estradiol on LH secretion.

The influence of dietary sunflower oil, rich in n-6 polyunsaturated fatty acids, in combination with vitamin C on ram semen parameters, sperm lipids and fertility.

Dietary fats, fed to ruminant animals, have been observed to improve reproductive function. Sunflower oil is one of the richest sources of polyunsaturated fatty acids (PUFAs), over 60% of which is linoleic acid (C18:2). With regard to recommendations for increased consumption of PUFAs, special emphasis has been given to the importance of consuming more antioxidants to prevent lipid peroxide formation. This study evaluated the effects of dietary n-6 PUFAs and vitamin C (VC) in rams-in particular, on semen quality, blood metabolites, sperm lipids and overall fertility. Diets supplemented with SFO and VC increased the proportion of motile sperms and their progressive motility improved (P < 0.01). Rams fed a diet containing SFO had an increased proportion of sperms with normal acrosomes in their semen samples (P < 0.01). The highest levels of lactate dehydrogenase activity (LDH) enzyme were found in control rams (2.60 U mL-1 ). Feeding SFO significantly affected blood triglyceride and cholesterol concentrations (P < 0.05). Diets containing SFO increased the proportion of C18:2c, C20:4, C20:3 and C22 in the sperm lipid composition (P < 0.05). Semen of SFO and VC-fed rams was more fertile than that of control rams when it was artificially inseminated to ewes. Feeding rams with supplemental SFO and VC increased semen quality and improved fertility rates. © 2019 Society of Chemical Industry.