Mature Rams Research Articles

Reproductive Response of Hair Sheep to Short or Long Duration Estrus Synchronization Protocols

Objective: Evaluate the reproductive response of hair ewes treated with estrus synchronization protocols that included a progesterone-releasing intravaginal device (CIDR) for a period of 7 or 12 days. Methodology: During the end of winter, ewes were randomly allotted to one of two estrus synchronization protocols: 1) Short-term protocol (SD), ewes (n=24) received a CIDR on Day 1 and removed on Day 7, and received 400 IU of equine chorionic gonadotropin (eCG) and 0.125 mg of cloprostenol; 2) Long-term protocol (LD), ewes (n=24) received a CIDR on Day 1, retired on Day 12 and received 400 IU eCG. Ewes showing estrous behavior were bred with a mature ram. Results: There were no differences (P>0.05) in the estrus response in both groups of ewes (100 vs 95.8% for SD and LD, respectively), the pregnancy rate (87.5 vs 87.5%), prolificacy (1.95+0.1 vs 2.2+0.1 lambs/ewe), hours between CIDR removing and the beginning of estrus (34.4+8.1 vs 34.8+9.2 h), and estrus duration (39.7+2.9 vs 39.9+4.7 h). At CIDR removal, ewes in the PL group showed a greater diameter (P<0.05) for the largest follicle in both ovaries (5.42+0.3 and 5.42+0.4 mm for the follicle in the left and right ovaries, respectively) vs PC ewes (4.25+0.1 and 4.29+0.1 mm). Limitations of study: Results observed in the follicle diameter could be due to low circulating progesterone levels in the LP group, which must be confirmed in future studies. Conclusions: The short protocol for estrus synchronization can be used in hair ewes under temperate conditions, without affecting their reproductive response.

PSLBI-13 Characterizing the seminal microbiota in mature rams managed on divergent planes of nutrition, and in their male offspring

Abstract Increasing evidence suggests that the bovine semen harbors commensal microbiota, and it may be important in male fertility. However, much is unknown regarding the seminal microbiota, its evolution and factors shaping this community in sheep. The objective of this study was to evaluate whether managing the rams on divergent planes of nutrition can influence the seminal microbiota in mature rams and their male offspring. For this, Rambouillet rams (n = 24; 1.5 to 4 yr old) were randomly assigned to one of three groups: 1) a positive plane of nutrition [target 12% gain in their initial body weight (BW; POS, n = 8); 2) a maintenance [maintain the initial BW (MNT, n = 8)]; and 3) a negative [target a 12% reduction in BW (NEG; n = 8)] plane of nutrition over 84 d. The rams were individually housed and fed a common diet, and feed allocations were adjusted weekly. Following the 84-d feeding period, these rams were used to breed 240 mature Rambouillet ewes (1:10 ram:ewe ratio) over 28 d of breeding. After lambing, the male lambs were maintained on a similar diet for 124 d. Semen samples from the mature rams (F0 generation) were collected on d 28, 56, and 84, and semen samples from offspring rams (F1 generation) were collected at 330 d of age via electroejaculation. Genomic DNA was extracted from semen samples and the seminal microbiota characterized using 16S rRNA gene (V3-V4) sequencing. Several environmental and negative extraction controls were also sequenced to assess potential contaminants in these samples. Overall community structure of the seminal microbiota in F0 rams was not affected by the divergent planes of nutrition (PERMANOVA: P ≥ 0.05) or by sampling time (P > 0.05). Microbial richness (observed ASVs) was also not different (P > 0.05) between the three groups of F0 rams at any of the sampling time points; however, microbial diversity (P < 0.05) in the F0 rams was influenced by sire dietary treatment. Microbial community structure, richness and diversity did not differ among the POS, MNT and NEG F1 generation rams (P > 0.05). Actinobacteriota, Firmicutes, Bacteroidota, Proteobacteria and Fusobacteriota were the most abundant phyla. Only Bacteroidota abundance was significantly (P < 0.05) different between three F0 groups, with POS rams had greater (18.6%) of this phylum on d 28 as compared with MNT (2.1%) and NEG (5.1%). The predominant genera identified included Fastidiosipila, Corynebacterium, Trueperella, Arthrobacter, Dietzia, Bifidobacterium, Streptobacillus, Ornithinimicrobium, and Porphyromonas, and their relative abundance was not affected (P > 0.05) by the dietary treatment in F0 rams. Overall, rams managed on positive and negative planes of nutrition harbored similar seminal microbiota to control rams, and divergent nutrition also had no effect on the offspring ram seminal microbiota.

The influence of the conditioned environment of sheep mesenchymal stromal cells on the qualitative parameters of sperm cells

The purpose of this research was to study the influence of the conditioned medium of mesenchymal stem cells from adipose tissue and bone marrow of sheep on the quality parameters of sperm during a threehour incubation. The sperm of sexually mature rams of the Romanov and Dorper breeds at the age of 1-2.5 years was taken and examined. A total of 10 samples were taken (n=10). Conditioned medium (CM) was collected after 72 h of cultivation in mattresses with 90-100% cell monolayer. Three groups of samples, 10 in each, were formed: control (spermatozoa 7x1010 cells/ml + PBS buffer 100 μl, pH – 7.4), first experimental (spermatozoa 7x1010 cells/ml + conditioned medium (CM) MSC BM 2x108 cells/ml, pH – 7.2), the second experimental one (spermatozoa 7x1010 cells/ml + conditioned medium (CM) MSCs in adipose tissue 2x108 cells/ml, pH – 7.2). Comparisons of values were made at each incubation stage (0, 1, 2 and 3 hours) within each group. It was found that the use of conditioned medium of mesenchymal stem cells from adipose tissue and bone marrow of sheep at a concentration of 2x108 cells/ml and spermatozoa at a concentration of 7x1010 cells/ml in terms of improving the quality indicators of germ cells. During a three-hour incubation of sheep sperm with conditioned medium of mesenchymal stem cells (MS), a decrease in the number of progressively moving sperm was observed. At the same time, a statistically significant difference was recorded in the first experimental group (CS MSC BM) on the 3rd hour of incubation: 1.9 times (p<0.01), as well as in the second experimental group (CS MSC VT) on the 2nd and 3rd hour of incubation compared with indicators of 0 hour of incubation: 1.4 times (p≤0.05) and 2.4 (p<0.01), respectively. A statistically significant decrease in the number of morphologically normal sperm was recorded during an hour-long incubation with conditioned medium of MSCs from sheep bone marrow and amounted to 43.90±2.22% (p=0.04).

Sperm long non-coding RNAs as markers for ram fertility.

It is critical in sheep farming to accurately estimate ram fertility for maintaining reproductive effectiveness and for production profitability. However, there is currently a lack of reliable biomarkers to estimate semen quality and ram fertility, which is hindering advances in animal science and technology. The objective of this study was to uncover long non-coding RNAs (lncRNAs) in sperm from rams with distinct fertility phenotypes. Mature rams were allocated into two groups: high and low fertility (HF; n = 31; 94.5 ± 2.8%, LF; n = 25; 83.1 ± 5.73%; P = 0.028) according to the pregnancy rates sired by the rams (average pregnancy rate; 89.4 ± 7.2%). Total RNAs were isolated from sperm of the highest- and lowest-fertility rams (n = 4, pregnancy rate; 99.2 ± 1.6%, and 73.6 ± 4.4%, respectively) followed by next-generation sequencing of the transcripts. We uncovered 11,209 lncRNAs from the sperm of rams with HF and LF. In comparison to each other, there were 93 differentially expressed (DE) lncRNAs in sperm from the two distinct fertility phenotypes. Of these, 141 mRNAs were upregulated and 134 were downregulated between HF and LF, respectively. Genes commonly enriched for 9 + 2 motile cilium and sperm flagellum were ABHD2, AK1, CABS1, ROPN1, SEPTIN2, SLIRP, and TEKT3. Moreover, CABS1, CCDC39, CFAP97D1, ROPN1, SLIRP, TEKT3, and TTC12 were commonly enriched in flagellated sperm motility and sperm motility. Differentially expressed mRNAs were enriched in the top 16 KEGG pathways. Targets of the differentially expressed lncRNAs elucidate functions in cis and trans manner using the genetic context of the lncRNA locus, and lncRNA sequences revealed 471 mRNAs targets of 10 lncRNAs. This study illustrates the existence of potential lncRNA biomarkers that can be implemented in analyzing the quality of ram sperm and determining the sperm fertility and is used in breeding soundness exams for precision livestock farming to ensure food security on a global scale.

Oral supplementation with Scabiosa artropurperea L. aqueous extract improves the invivo sexual behaviour, sperm quality and androgen level in rams.

The effects of an aqueous extract of Scabiosa atropurpurea L. (AES) on the reproduction potential of Queue Fine de l'Ouest rams were evaluated over 9 weeks. Eighteen mature (4-6 years old) rams (52.8 ± 2.6 kg) were divided into three groups. The control (C) group was fed oat hay adlibitum with 700 g of concentrate and the other two groups were fed the same diet supplemented with AES at 1 and 2 mg/kg body weight (AES1 and AES2, respectively). Ram sperm was collected with an artificial vagina (2 × 2 days/week) to evaluate sperm production and quality, antioxidant activity, the adenosine triphosphate (ATP) and calcium concentrations. Sexual behaviour and plasma testosterone concentrations were also investigated. The administration of AES improved sexual behaviour (the duration of contact and the number of lateral approaches). The addition of AES also improved individual spermatozoa motility (C: 71.7% ± 6.3%; AES1: 78.3% ± 4.9%; AES2: 83.8% ± 4.4%), the sperm concentration (C: 5.6 ± 0.36; AES1: 6.4 ± 0.81; AES2: 6.7 ± 0.52 × 109 spermatozoa/mL), the ATP ratio (C: 1 ± 0.08; AES1: 2.1 ± 0.08; AES2: 3.3 ± 0.08) and the calcium concentration (C: 5.6 ± 0.24; AES1: 7.7 ± 0.21; AES2: 8.1 ± 0.24 mmol/L). AES treatment decreased the percentage of abnormal sperm (C: 18.5% ± 1.2%; AES1: 16.2% ± 1.1%; AES2: 14.8% ± 0.94%) and DNA damage (C: 62%; AES1: 27%; AES2: 33%) and was associated with elevated seminal fluid antioxidant activity (C: 22 ± 0.27; AES1: 27.1 ± 1.08 and AES2: 27.5 ± 0.36 mmol Trolox equivalents/L) and plasma testosterone (C: 8.3 ± 0.7; AES1: 11.7 ± 0.4; AES2: 15 ± 0.7 ng/L). In conclusion, our study suggests that S. atropurpurea may be potentially useful to enhance libido and sperm production and quality in ram.

Effect of dietary zinc, selenium and their combination on antioxidant parameters in serum and Semen of Sanjabi mature rams

BackgroundSelenium and zinc play as antioxidants in biological systems, playing important roles in spermatogenesis and protecting against oxidative damage caused by oxygen radicals. This study aimed to investigate the effects of dietary intake of zinc and selenium, either separately or in combination, on antioxidant parameters in the blood and semen of ram. MethodsTwenty mature fat-tailed Sanjabi rams, weighing 50.01 ± 5.6 kg and 20 months of age, were randomly assigned to four different treatments for a period of 120 days (from July to November). The experimental groups included: 1) the basal diet (control group, with 0.11 mg.kg−1 selenium and 20.52 mg.kg−1 zinc) (C), 2) the basal diet supplemented with 0.3 mg.kg−1 selenium (Se), 3) the basal diet supplemented with 40 mg.kg−1 zinc (Zn), and 4) the basal diet supplemented with both selenium (0.3 mg.kg−1) and zinc (40 mg.kg−1) (ZS). Blood samples were collected at eight-week intervals for analysis. Specifically, the levels of total antioxidant status (TAS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and zinc (Zn) in serum, as well as GSH-Px, Zn and MDA concentrations in semen were measured. ResultsDuring the first two months of the experiment, the serum total antioxidant status (TAS) in all of the treated animals was significantly higher than that of the control group (P<0.01). However, after the second month, only the rams in the selenium (Se) group continued to have a significantly greater serum TAS compared to the controls until the fourth month of the experiment (P< 0.05). There was no significant difference in malondialdehyde (MDA) levels in the serum of treated animals compared to the controls at any point during the 120-day experiment (P >0.05). The serum glutathione peroxidase (GSH-Px) activity was significantly higher in the selenium (Se) group compared to the other groups (P <0.01). However, in the seminal plasma, the GSH-Px activity was found to be increased in rams supplemented with both zinc (Zn) and Se (ZS group) compared to the controls on days 60 and 120 of the experiment (P <0.05). Similarly, the dietary supplementation of selenium (Se) and zinc (Zn) resulted in a significant increase in both the seminal and blood serum zinc concentration in fat-tailed Sanjabi rams on day 120 when compared to the control group. ConclusionsThe dietary supplementation of zinc (Zn) and selenium (Se) was shown to increase the antioxidant levels in both the seminal plasma and blood serum of mature Sanjabi rams. This enhancement in antioxidant status may potentially lead to an improvement in the reproductive performance of these treated rams during the breeding season.

Divergent planes of nutrition in mature rams influences body composition, hormone and metabolite concentrations, and offspring birth measurements, but not semen characteristics or offspring growth.

Objectives of this experiment were to characterize the effects of ram plane of nutrition on body composition, concentrations of hormones and metabolites, sperm characteristics, and offspring outcomes. Mature Rambouillet rams (n = 24, BW = 82.9 ± 2.63kg) were individually housed and randomly assigned to either a positive (POS; n = 8), maintenance (MAINT; n = 8), or negative (NEG; n = 8) plane of nutrition for an 84-day feeding period. Rams were fed a common diet, with daily feed allocations adjusted weekly based on body weight (BW) to achieve the targeted weight gain or loss (approximately 12% of initial BW). On 0, 28, 56, and 84-d, body condition score (BCS) and scrotal circumference (SC) were recorded, and blood and semen were collected. Following the feeding period, rams were placed in pens with 10 ewes each for a 28-d breeding period. Ewes were managed similarly throughout gestation and body weight and measurements were recorded at birth and weaning. Data were analyzed as repeated measures in time where appropriate with the mixed procedure of SAS, and individual ram was the experimental unit for all analysis. Ram BW was influenced by a treatment × day interaction (P < 0.001), with POS (0.12 ± 0.01kg) having greater daily weight change than MAINT (0.1 ± 0.01kg), which was greater than NEG (-0.12 ± 0.01kg). Ram BCS and SC were influenced by treatment × day interactions (P ≤ 0.01), being similar on day 0 but POS being greater than NEG by day 56. Concentrations of triiodothyronine (T3) and T3:T4 ratio exhibited treatment × day interactions (P ≤ 0.02), as POS had greater values than NEG by day 84 (P ≤ 0.02). Concentration of insulin-like growth factor-1 was greater in POS than MAINT and NEG (P ≤ 0.02), and non-esterified fatty acids and thyroxine (T4) were influenced by a day effect (P ≤ 0.01), but testosterone was unaffected (P ≥ 0.09). Minimal differences in semen volume, sperm concentration, motility, or morphology were observed among treatments (P ≥ 0.31). A similar proportion of ewes bred by rams in the respective treatments lambed and weaned lambs (P ≥ 0.54). Birth weight, chest circumference, and shoulder-hip length were greater (P ≤ 0.05) in NEG lambs compared with POS and MAINT; however, no differences were detected in weaning weight and weaning body measurements (P ≥ 0.40). Findings suggest paternal nutrition during the period of sperm development may influence offspring outcomes, potentially as a result of in-utero programming of paternal origin.

Genetic impact of external Targhee sires at the U.S. Sheep Experiment Station: a case study of introgression.

Sheep breeders requested that the U.S. Sheep Experiment Station (USSES) to participate in national genetic evaluation through the National Sheep Improvement Program (NSIP). The reasons included the need for (1) a comparison of the productivity of industry and United States Department of Agriculture (USDA) lines, (2) transparency of USDA flocks, (3) genetic ties for NSIP by sampling of industry flocks, and (4) development of premium genetic lines for public release. In response, USSES began to incorporate external sires from NSIP participating flocks into the USSES Targhee flock. Our objective, based on a pedigree analysis, was to test if introgression of external genetics into the flock was achieved. The pedigree included 13,189 animals with mean maximum generations, mean complete generations, and mean equivalent complete generations of 4.2, 1.8, and 2.6, respectively. The mean generation interval was 3.1 yr. The reference population was defined as lambs born from 2021 to 2023 (n = 792). Two additional populations were defined as the current mature ewe flock (n = 123) and the current mature rams (n = 14). The Genetic Conservation Index averaged 7.7 for the full population and 25.7 for the reference population. Overall inbreeding was 0.003 for the full population and 0.006 for the reference population. The rate of inbreeding was 0.0003 per generation. Average relatedness was 0.015 for the full population and 0.018 for the reference population. The effective number of founders, effective number of ancestors, and founder genome equivalents contributing to the reference population were 60, 39, and 19.1, respectively. The ratio of the effective number of founders to the effective number of ancestors was 1.5, indicating the presence of genetic bottlenecks. Measures of effective population size ranged from 102 to 547. Of the 704 offspring produced by external sires, 17 ram lambs and 132 ewe lambs were retained for breeding. The USSES sires produced 299 offspring with 2 ram lambs and 51 ewe lambs retained. Incorporating external sires resulted in a cumulative percentage of genetic variance of 48.8, 49.1, and 44.2 of external genetics for the reference population, current mature ewe flock, and current mature rams, respectively. Stakeholder needs were addressed by introgression of external sires and participation in NSIP, but future selection practices need to be modified to maintain a minimum of 50% USSES core genetics in the flock.

Effect of acetic acid addition to rice straw pre-treated with urea on performance of dairy ewes.

This experiment was carried out to study the effect of acetic acid addition to rice straws that had been already treated with urea on chemical composition, digestibility, nutritive value, and productive performance of ewes. Thirty ewes with an initial body weight of 44 ± 0.41 kg were chosen 7 days after parturition and divided into 3 groups (10 of each) for 3 months. Each group was assigned randomly to receive one of the three experimental rations composed of concentrate feed mixture and untreated rice straw as a control group (G1), urea-treated rice straw (G2), and urea-acetic acid-treated rice straw (G3). Digestibility coefficients and nutritive value were determined using fifteen mature rams andblood samples were obtained at the end of the collecting period. Findings indicated that the crude protein of rice straw was increased, and crude fiber content was decreased by either urea or urea-acetic acid rations(i.e., G2 and G3). Digestibility of most nutrients was increased (P < 0.05) for animals fed either G2 or G3 rations. There were no significant differences in nutritive value astotal digestible nutrients (TDN) among tested groups; however, digestible crude protein(DCP) increased (P < 0.05) for G2 and G3 rations. Plasma total protein, AST, creatinine, and urea were increased (P < 0.05) by feeding on G2 or G3 ration; however, plasma ALT was not affected. The average daily intake ofdry matter (DM), TDN, and DCP was higher for G2 and G3 rations. Actual milk yield, fat-corrected milk(FCM), percentages of fat, and total solids and constituents yield were increased (P < 0.05) by feeding on treated rice straw rations (i.e.,G2 and G3); however, the percentages of protein, lactose, solids not fat, and ash were not affected. In the 1st month after parturition, the changes in ewes body weight were less (P < 0.05) when they fed on G2 and G3 rations than the control one(G1)). Dams' production and offspring performance were improved (P < 0.05) by feeding on G2 and G3 rations. Feed conversion as kg DMI/kg FCM, kg TDN/kg FCM, and economic efficiency was improved for G2 and G3. Inconcluded, adding acetic acid to urea-treated rice straw may improve the performance of ewes fed on it and increase their milk production and profitability. The same trend was observed with (G2) which received urea-treated rice straw ration.

Minimizing sperm oxidative stress using nanotechnology for breeding programs in rams

BackgroundArtificial insemination (AI) is a routine breeding technology in animal reproduction. Nevertheless, the temperature-sensitive nature and short fertile lifespan of ram sperm samples hamper its use in AI. In this sense, nanotechnology is an interesting tool to improve sperm protection due to the development of nanomaterials for AI, which could be used as delivery vehicles. In this work, we explored the feasibility of vitamin E nanoemulsion (NE) for improving sperm quality during transport.ResultsWith the aim of evaluating this proposal, ejaculates of 7 mature rams of Manchega breed were collected by artificial vagina and extended to 60 × 106 spz/mL in Andromed®. Samples containing control and NE (12 mmol/L) with and without exogenous oxidative stress (100 µmol/L Fe2+/ascorbate) were stored at 22 and 15 ºC and motility (CASA), viability (YO-PRO/PI), acrosomal integrity (PNA-FITC/PI), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) monitored during 96 h. Our results show that NE could be used to maintain ram spermatozoa during transport at 15 and 22 ºC for up to 96 h, with no appreciable loss of kinematic and physiological characteristics of freshly collected samples.ConclusionsThe storage of ram spermatozoa in liquid form for 2–5 d with vitamin E nanoemulsions may lead more flexibility to breeders in AI programs. In view of the potential and high versatility of these nanodevices, further studies are being carried out to assess the proposed sperm preservation medium on fertility after artificial insemination.Graphical

PSII-7 Feed Intake and Behavioral Responses of Mature Rams Provided Feed Containing Phenylthiocarbamide

Abstract Bitter taste perception has been studied in various species, including humans, using the synthetic compound phenylthiocarbamide (PTC). Human perception of PTC is linked to the ability to taste bitter foods. Sheep express varying levels of aversion to PTC delivered in drinking water. The objective of this study was to determine the effects of PTC delivered in feed on dietary intake and behavior in mature rams. We hypothesized that rams previously classified into PTC-taster categories using PTC-spiked drinking water would rank similarly when PTC was added to feed. Mature Targhee, Rambouillet, Polypay, and composite-breed rams, previously classified as either super- (n = 3), intermediate- (n = 5), or non-PTC tasters (n = 4), were subjected to 4-d acclimation and 6-d test phases. Alfalfa (70%), corn (15%), and beat pulp (15%) meal were mixed with ethanol (150 g/kg, as fed) only (control) or PTC solubilized in ethanol (150 g/kg, as fed) to create three pelleted (6.3 mm) test diets (110, 220 and 330 mg PTC/kg feed, as fed). Rams received control and PTC diets (0.87% BW) daily for 30 min in a side-by-side presentation, which was replicated for each PTC concentration. Feeding duration, number of bucket approaches and switches, sniffs, drinks, lip smacks and licks, and head bobs, jerks, and shakes were recorded throughout the trial using video surveillance. For each PTC concentration, consumption of PTC and control diets was similar (P = 0.22) when measured as a percentage of total diet (control + PTC) consumed. Individual PTC diet intakes ranged from 1.5 to 89.8% of total diet consumed. The CV for intake of 110, 220 and 330 mg/kg diets increased from 22.4 to 34.1 to 36.1%, respectively. Intake of total diet offered decreased (P = 0.01) between 110 mg/kg (80.9 ± 3.2%) and 330 mg/kg (72.3 ± 3.2%), while 220 mg/kg (78.4 ± 3.2%) was intermediate (P ≥ 0.06). Rams were classified by average PTC diet consumption relative to ± 1.0 SD of the population mean (50.2 ± 7.5%). Using this method, only 7 of the 12 rams were classified similarly to the former classification established when PTC was added to drinking water. Total feeding duration was less (P ≤ 0.03) when rams were offered 330 mg/kg (805.4 ± 18.2 s) compared with 110 (858.1 ± 19.3 s) and 220 mg/kg (862.7 ± 19.5 s). Rams switched between the control and PTC diets more (P ≤ 0.02) when offered 110 mg/kg (21.2 ± 10.0) than the 220 (16.4 ± 7.8) and 330 mg/kg (18.4 ± 8.4). These results suggest that sheep perceive and exhibit variable responses to PTC when incorporated into a pelleted feed. More research is needed to identify mechanisms by which sheep perceive bitter-tasting feeds.

Vitamin E Lipid-Based Nanodevices as a Tool for Ovine Sperm Protection against Oxidative Stress: Impact on Sperm Motility.

The advent of nanotechnology in the field of animal reproduction has led to the development of safer and more efficient therapies. The use of nanotechnology allows us to avoid the detrimental effects of certain traditional antioxidants, such as Vitamin E. Its hydrophobic nature makes mandatory the use of organic solvents, which are toxic to sperm cells. This study aims to evaluate the efficiency of vitamin E nanoemulsions (NE) on ram (Ovis aries) spermatozoa. For this purpose, the effect of three NE concentrations (6, 12, and 24 mM) were assessed on sperm of 10 mature rams of the Manchega breed. Sperm samples were collected by artificial vagina, pooled, and diluted in Bovine Gamete Medium. The samples were stored at 37 °C and assessed at 0, 4, 8, and 24 h under oxidative stress conditions (100 µM Fe2+/ascorbate). Motility (CASA), viability (YO-PRO/IP), acrosomal integrity (PNA-FITC/IP), mitochondrial membrane potential (Mitotracker Deep Red 633), lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®®) were assessed. A linear mixed-effects models were used to analyze the effects of time, NE, and oxidant (fixed factors) on sperm parameters, and a random effect on the male was also included in the model with Tukey’s post hoc test. Protection of ram spermatozoa with NE resulted in a more vigorous motility under oxidative stress conditions with respect Control and Free vitamin E, while preventing the deleterious effects of oxidative stress coming from the production of free radicals and lipid peroxidation. These results ascertain the high relevance of the use of delivery systems for sperm physiology preservation in the context of assisted reproduction techniques.

Refined methodology for identification of bitterness aversion in mature rams through quantification of fluid intake and behavioral response to phenylthiocarbamide

Sheep have a sensitive threshold for bitterness, which may affect feed selection. Previous research attempted to classify sheep according by bitter sensitivity by measuring fluid intake of the compound phenylthiocarbamide (PTC) at various concentrations over a 14-d period. Although successful, the study was laborious, lengthy, required multiple layers of acclimation and testing, and accommodated a limited number of animals. Accordingly, a more efficient test is needed to classify bitter-taster status of sheep. The objective of this experiment was to develop a more rapid and refined method to assist in identification of PTC-taster status in rams utilizing only two concentrations of PTC-mixture over a 2-day test period. Mature Targhee, Rambouillet, Polypay, and composite-breed rams (n = 44), were equally stratified into three barns and subjected to 4-d acclimation, 2-d sham, and 2-d testing phases. The sham phase was conducted to determine if ethanol (delivery vehicle for PTC) influenced voluntary fluid intake or behavior; no effect (P ≥ 0.13) was observed. The 2-d test phase was a side-by-side preference study between water and one of two concentrations (0.20 and 2.03 mM) of PTC-mixture delivered on alternate days. Video surveillance was conducted to evaluate the number and duration of approaches to the PTC-mixture and subsequent sniffing, head bobbing, jerking, shaking, and lip movements, smacking, and licking behaviors. Overall, intake of PTC-mixture was less (P < 0.001) than water intake. The PTC-taster classifications were determined based on average PTC intake relative to ± 1.0 standard deviation of the study population mean and used to sort rams into super- (n = 9), intermediate (n = 29), or non- (n = 6) PTC-taster classifications. The amount of time spent drinking either PTC-mixture or water was different (P < 0.001) amongst PTC-taster classifications and correlated with both water (r = 0.23, P = 0.028) and PTC-mixture (r = 0.46, P < 0.001) intake. Head bobbing (P = 0.035) and lip smacking (P < 0.001) were associated with PTC intake across all classes. Super-tasters appeared to utilize sense of smell (sniffing) more frequently to identify PTC-mixture than the intermediate or non-tasters (P ≤ 0.012). Non-tasters did not exhibit behavioral differences (P ≥ 0.12) regardless of solution type. The streamlined methodology outlined in this study will help facilitate future studies aimed at better understanding forage selection and consumption in sheep.

Morphological and ultrasonographic characterization of the three zones of supratesticular region of testicular artery in Assaf rams

To fully understand the histological, morphometrical and heamodynamic variations of different supratesticular artery regions, 20 mature and healthy Assaf rams were examined through ultrasound and morphological studies. The testicular artery images of the spermatic cord as shown by B-mode analysis indicated a tortuous pattern along its course toward the testis, although it tends to be less tortuous close to the inguinal ring. Doppler velocimetric values showed a progressive decline in flow velocity, in addition to pulsatility and vessel resistivity when entering the testis, where there were significant differences in the Doppler indices and velocities among the different regions. The peak systolic velocity, pulsatility index and resistive index were higher in the proximal supratesticular artery region, followed by middle and distal ones, while the end diastolic velocity was higher in the distal supratesticular region. The total arterial blood flow and total arterial blood flow rate reported a progressive and significant increase along the testicular cord until entering the testis. Histological examination revealed presence of vasa vasorum in the tunica adventitia, with their diameter is higher in the proximal supratesticular zone than middle and distal ones. Morphometrically, the thickness of the supratesticular artery wall showed a significant decline downward toward the testis; meanwhile, the outer arterial diameter and inner luminal diameter displayed a significant increase distally. The expression of alpha smooth muscle actin and vimentin was higher in the tunica media of the proximal supratesticular artery zone than in middle and distal ones.

59 Divergent Planes of Nutrition Altered Concentrations of Hormones and Metabolites but not Semen Characteristics in Mature Rams

Abstract Objectives were to evaluate impacts of divergent planes of nutrition on body weight (BW), hormone and metabolite concentrations, and semen characteristics in rams. Mature Rambouillet rams (n = 24, BW = 83.1 ± 2.64 kg) were individually housed and randomly assigned to positive (POS; n = 8), maintenance (MAINT; n = 8), or negative (NEG; n = 8) planes of nutrition for 84 d. Rams were fed a common diet with feed allocations adjusted weekly based on BW to achieve targeted weight gain or loss (approximately 12% initial BW). On d 0, 28, 56, and 84, body condition scores (BCS) and scrotal circumference (SC) were recorded, and blood and semen were collected. Blood was analyzed for triiodothyronine (T3), thyroxine (T4), testosterone, insulin-like growth factor-1 (IGF-1), glucose, and non-esterified fatty acids (NEFA). Semen was collected via electroejaculation and analyzed by Computer Assisted Semen Analysis. Data were analyzed with the GLM or MIXED procedure of SAS, with ram as experimental unit and significance determined at P &amp;lt; 0.05. By design, BW was influenced by a treatment × day interaction (P &amp;lt; 0.0001), and daily weight change was greater (P &amp;lt; 0.0001) for POS (0.11 ± 0.011 kg) than MAINT (0.01 ± 0.011 kg), which was greater (P &amp;lt; 0.0001) than NEG (-0.12 ± 0.011 kg). Consequently, BCS, and SC were greater for POS than NEG as evidenced by a treatment × day interaction (P &amp;lt; 0.0001). Concentrations of T3, IGF-1, and NEFA were increased in POS compared with NEG and MAINT by day 84 (P ≤ 0.03), whereas T4, testosterone, and glucose remained unaffected (P ≤ 0.39). No differences were observed among treatments for semen volume/concentration, motility, or morphology (P ≤ 0.49). Collectively, BW, hormone, and metabolites differed among treatments, indicating a potential for epigenetic alterations which could influence offspring outcomes.

Biometric parameters and computer assisted analysis of pixel distribution on B-mode testicular ultrasonogram in young and sexually mature rams

This study was designed to investigate the relationships between basic biometric parameters and pixel distribution in predetermined regions of B-mode testicular ultrasonogram in young and sexually mature rams by computer assisted analysis. The animals were separated in two groups: group I (young rams, aged 5-8 months; n=6) and group II (sexually mature rams, aged 14-18 months; n=6). Biometric parameters as age, body weight and scrotal circumference were determined by routine methods. A trans-scrotal ultrasonography of both testes was performed with 7 MHz linear transrectal probe by the same operator with focus, gain and brightness values kept constant during the study. The ultrasound images were obtained in a longitudinal view plane and frozen when visualisation of the testicular mediastinum was clear and apparent. All images were transferred to a computer, converted to gray-scale (0-255 pixels) and submitted to pixel distribution analysis using Image ProPlus 7.0 analytical software and option range statistics. The pixel distribution analysis in the same region for left and right testes was based on the pixel ranges classification for different biological tissues (blood, lipids, muscle, fibrous and calcium) and expressed as colour area in percentages. The arithmetic mean of values for the left and right testis was accepted as a final value for each ram. The data for both groups were statistically processed, compared and the relationships between the biometric parameters and different pixel ranges were determined. Significant differences (P&lt;0.05) among the groups were recorded for all biometric parameters and the lipid area only. The biometric parameters correlated positively (R≥0.92; P&lt;0.05) each with the other, while their relationship (R≤ -0.94; P&lt;0.05) with the lipid area and the correlation between muscle and fibrous area were negative (R= -0.92; P&lt;0.05). In conclusion, the pixel distribution analysis of a predetermined region of B-mode testicular ultrasonogram based on pixel ranges classification for different biological tissues can be used for detection of testicular parenchyma changes in rams. Future comparative histologic and ultrasound investigations are needed to determine correct pixel values for different biological tissues in the testes of young and sexually mature rams.

Chios ram testicular blood flow and echotexture changes depending on age, season and ejaculation process.

The present study aimed to investigate the impact of age, season and ejaculation on ram testicular blood flow and echotexture. The survey was conducted biweekly on 7 Chios rams for one year, including breeding and non-breeding periods. The rams were divided into 2 age groups: 3 rams 2-6 years old (mature) and 4 rams 9-13 years old (old). Hemodynamic indices [Pulsatility index (PI), Resistive index (RI), End-diastolic velocity (EDV), testicular artery Diameter (D), Time-averaged maximum velocity (TAVM), Blood flow volume (BFV)] and echotexture parameters [Mean value (MV), Contrast (Con), Gray value distribution (GVD), Run length distribution (RunLD), Long run emphasis (LRunEm), Entropy (Ent), Correlation (Cor), Standard deviation (StD), Gray variance (GV) and Gradient mean value (GMV)] were evaluated in each testis before and after ejaculation. Ejaculation did not affect testes blood flow or echotexture (p>0.05). PI and RI were higher in the breeding period compared to the non-breeding period, for both testes (p⟨0.001). Left testis GV and Cor before ejaculation were lower (p=0.01) and higher (p=0.03), respectively, in the breeding compared to the non-breeding period. Left testis D (p=0.005) and BFV (p⟨0.001) were higher in old compared to mature rams after ejaculation. Right testis Con (p=0.03) and Cor (p=0.05) before ejaculation were higher in old rams, whereas right testis Ent after ejaculation was higher in mature rams (p=0.05). In conclusion, testicular blood flow and echotexture are affected by season and ram age, but not by ejaculation.

Proteomic fertility markers in ram sperm

Precise estimation of ram fertility is important for sheep farming to sustain reproduction efficiency and profitability of production. There, however, is no conventional method to accurately predict ram fertility. The objective of this study, therefore, was to ascertain proteomic profiles of ram sperm having contrasting fertility phenotypes. Mature rams (n = 66) having greater pregnancy rates than average (89.4 ± 7.2%) were assigned into relatively-greater fertility (GF; n = 31; 94.5 ± 2.8%) whereas those with less-than-average pregnancy rates were assigned into a lesser-fertility (LF; n = 25; 83.1 ± 5.73%; P = 0.028) group. Sperm samples from the outlier greatest- and least-fertility rams (n = 6, pregnancy rate; 98.4 ± 1.8% and 76.1 ± 3.9%) were used for proteomics assessments utilizing Label-free LC-MS/MS. A total of 997 proteins were identified, and among these, 840 were shared by both groups, and 57 and 93 were unique to GF and LF, respectively. Furthermore, 190 differentially abundant proteins were identified; the abundance of 124 was larger in GF while 66 was larger in LF rams. The GF ram sperm had 79 GO/pathway terms in ten major biological networks while there were 47 GO/pathway terms in six biological networks in sperm of LF rams. Accordingly, differential abundances of sperm proteins between sperm of GF and LF rams were indicative of functional implications of sperm proteome on male fertility. The results of this study emphasize there are potential protein markers for evaluation of semen quality and estimation of ram sperm fertilizing capacity.

361 Methods for Identification and Classification of Phenylthiocarbamide Sensitivity in Mature Rams

Abstract Rams exhibit varying levels of sensitivity to phenylthiocarbamide (PTC), which has been correlated with the ability to perceive bitter-tasting foods. Previously, the only procedure for measuring PTC-sensitivity in rams involved a 9-d test phase with 5 PTC concentrations. The objective of this experiment was to develop a more streamlined approach for identifying and classifying rams on extreme ends of the bitter-tasting spectrum. Mature Targhee, Rambouillet, Polypay, and Composite-breed rams (n = 44), housed in three separate barns, and subjected to 4-d acclimation, 2-d sham, and 2-d testing phases. The 2-d sham phase was conducted to determine if ethanol-spiked water (delivery vehicle for PTC) influenced voluntary fluid intake; no effect (P = 0.16) was observed. The 2-d test phase was a side-by-side preference study between water (control; 3.0 kg) and 1 of 2 concentrations (0.2 and 2.0 mM) of PTC solution (3.0 kg) delivered on alternate days. Average consumption of PTC as a percentage of total test fluid intake was used determine taster status. No PTC-treatment effect (P ≥ 0.23) was observed, but within each treatment level, PTC intake was less (P &amp;lt; 0.001) than water intake. Classification levels of PTC consumption were determined by ± 1.0 standard deviation of the mean and used to classify rams into non- (n = 9; 61.5 ± 3.9%), intermediate- (n = 29; 40.7 ± 1.8%), or super- (n = 6; 15.6 ± 3.3%) PTC tasting groups, which were different (P &amp;lt; 0.001) from each other. Likewise, water consumption intakes were different (P&amp;lt; 0.001) between the non- (38.5 ± 3.9%), intermediate- (59.3 ± 1.8%), and super- (84.4 ± 3.3%) PTC tasting groups. The methods outlined in this study will facilitate further research focused on the influence of bitter tasting sensitivities on the dietary preferences of sheep.

Effect of Different Pre-Freezing Time on Quality of Frozen Fat-Tailed Ram Semen

This study aimed to determining the effect of temperature changes on the sperm quality of fat-tailed sheep during the freezing process using a microcontroller. This study was conducted from April to October 2018 at the Laboratory of Animal Physiology and Reproduction, Faculty of Animal Science UGM. The study was used twelve mature ram. The methods were conducted by collecting semen using artificial vagina, semen dilution, freezing semen, thawing and semen quality test. The pre-freezing temperature time were grouped for 6, 9, and 12 minutes. Data of frozen semen quality (motility, viability, and abnormality) the data were analyzed using one-way ANOVA and the study was arranged using Completely Randomized Design (CRD). The average of spermatozoa motility after semen freezing with a 5 to -140°C decrease in pre freezing temperature for 12 minutes (50±5.3%) was significantly different (P&lt;0.05) compared to 9 minutes (48±4.8%) and 6 minutes (43±4.8%). The average of spermatozoa viability after semen freezing with a decrease in pre freezing temperature of 5 to -140°C for 12 minutes (55±4.7%) was significantly different (P&lt;0.05) compared to that for 9 minutes (52±3.5%) and 6 minutes (49±5.7%). The average of spermatozoa abnormality after freezing with a decrease in pre freezing temperature of 5 to -140°C for 6, 9, and 12 minutes was not significantly different (10±2.4%, 9±0.8%, and 10±0.9%, respectively). Based on the findings, it is possible to conclude that semen freezing at a lower pre-freezing temperature of 0 to -140°C for 12 minutes can improve the quality of freezing results.