Scrotal subcutaneous temperature is increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decrease sperm quality in bulls and rams.

Ruminant testes are ~2-6 °C below body temperature; increased testicular temperature reduces sperm motility and morphology. Our objective was to serially monitor scrotal subcutaneous temperatures during testicular heat stress and relate those to sperm quality. Two experiments were conducted, with temperature sensors surgically implanted in scrotal subcutaneous tissues recording temperatures every 15 min and semen collected and evaluated weekly. After an initial control interval, testicular temperature was increased. In Experiment 1, in two Angus bulls, whole-scrotum insulation for 96 h increased scrotal subcutaneous temperatures by ~2.0-2.5 °C (P < 0.05). Total and progressive motility decreased (P < 0.05) and reached a nadir at Week 3 (~20 and 10%, respectively). Furthermore, morphologically normal sperm and acrosome integrity also decreased significantly, reaching nadirs at Weeks 3 (15%) and 4 (34%). In Experiment 2, 10 Dorset rams were allocated randomly into two equal groups and either: 1) exposed to 28 °C ambient temperature and 30-34% RH for 8 h/d for 4 d; or 2) subjected to scrotal neck insulation that was applied and removed at the same time as the start and completion, respectively, of heat exposures in the other rams. Scrotal subcutaneous temperatures (monitored in three rams per group) were increased in response to whole-body heating (~0.8 °C, P < 0.05), but not significantly changed by scrotal neck insulation. Decreases in sperm quality were generally similar between treatments, with the most profound changes evident 4 wk after heat stress, with ~10 percentage point reductions in both total and progressive motility and ~10 and 20 percentage point decreases in morphologically normal sperm in rams with whole-body heating versus scrotal neck insulation, respectively. In conclusion, scrotal subcutaneous temperature was significantly increased by scrotal insulation or whole-body heating, but not by scrotal neck insulation; however, all three heat-stress models decreased sperm motility and morphology in bulls and rams.

Effect of racial crossing on the seminal parameters of rams submitted to heat stress / Efeito do cruzamento racial nos parâmetros seminais de carneiros submetidos ao estresse de calor

The aim of this study was to evaluate the effect of racial crossing on seminal parameters of eight Santa Inês and crossbred (Santa Inês x Dorper) rams submitted to heat stress, and to monitor the return of these parameters to previously reported. Before to place the insulation bags, two collects of semen through electroejaculation were performed. The insulation pouches were made with double-layer plastic, internally lined with cotton, and fixed around the spermatic funiculus and scrotum with adhesive tape and bandage remaining on the testes of the animals for seven days. The first collect was performed on the day that the pouches were taken (day 0) and thereafter, every seven days, totalizing 15 measurements. Data were submitted to analysis of variance (ANOVA). The analyzed variables were subjected to Dunnett test at 5% probability to compare the values obtained before treatment with those obtained in the following days. In this study it was found that the animals restored normal seminal parameter after the insulation effects, however, the return rate differed slightly among the studied breeds. The crossbred animals restored the seminal patterns, on average, a week before Santa Inês. It is concluded that the racial crossing influences the semen parameters of rams submitted to heat stress.

O objetivo deste estudo foi avaliar o efeito do cruzamento racial sobre parâmetros seminais de oito carneiros Santa Inês e mestiços, submetidos ao estresse térmico e monitorar o retorno desses parâmetros aos relatos anteriormente. Antes de colocar as bolsas de insulação, foram realizadas duas coletas de sêmen por meio de eletroejaculação. As bolsas de insulação foram confeccionadas com plástico de camada dupla, revestidas internamente com algodão, fixadas ao redor do funículo espermático e escroto com fita adesiva e bandagem, permanecendo nos testículos dos animais por sete dias. A primeira coleta foi realizada no dia em que as bolsas foram retiradas (dia 0) e a partir daí, a cada sete dias, totalizando 15 coletas. Os dados foram submetidos à análise de variância (ANOVA). As variáveis analisadas foram submetidas ao teste de Dunnett a 5% de probabilidade para comparar os valores obtidos antes do tratamento com aqueles obtidos nos dias seguintes. Neste estudo verificou-se que os animais restauraram os parâmetros seminais normais após os efeitos da insulação, porém, a taxa de retorno diferiu ligeiramente entre as raças estudadas. Os animais mestiços restauraram os padrões seminais, em média, uma semana antes da Santa Inês. Conclui-se que o cruzamento racial influencia os parâmetros seminais de carneiros submetidos ao estresse térmico.

Effects of increased scrotal temperature on semen quality and seminal plasma proteins in Brahman bulls.

Environmental temperature has effects on sperm quality with differences in susceptibility between cattle subspecies and breeds, but very little is known about the seminal plasma protein (SPP) changes resulting from testicular heat stress. Scrotal insulation (SI) for 48 hr was applied to Brahman (Bos indicus) bulls. Semen was collected at 3-day intervals from before, until 74 days post-SI. The changes in sperm morphology and motility following SI were comparable to previously reported and differences were detected in measures of sperm chromatin conformation as early as 8 days post-SI. New proteins spots, in the SPP two-dimensional (2-D) gels, were apparent when comparing pre-SI with 74 days post-SI, and SPP identified as associated with mechanisms of cellular repair and protection. Similar trends between 2-D gel and Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) data was observed, with SWATH-MS able to quantify individual SPP that otherwise were not resolved on 2-D gel. The SPP assessment at peak sperm damage (21-24 days) showed a significant difference in 29 SPP (adjusted p < .05), and identified six proteins with change in abundance in the SI group. In conclusion both spermatozoa and SPP composition of bulls are susceptible to temperature change incurred by SI, and SPP markers for testicular heat insults may be detected.

Effect of scrotal insulation on sperm quality and seminal plasma proteome of Brangus bulls.

Seminal plasma (SP) contributes to sperm physiology and metabolism, prevents premature capacitation, and protects sperm against oxidative stress. In order to evaluate the impact of heat stress in the semen of tropically adapted Brangus breed and in their seminal plasma proteome, we studied the effects of scrotal insulation for 72 h. Semen samples from six bulls, between 7 and 8 years of age, were collected prior to scrotal insulation (pre-insulation), and at 4 and 11 wk after insulation. Seminal plasma samples were analyzed by 2D SDS-PAGE and liquid chromatography coupled with mass spectrometry (LC-MS/MS). Insulation caused decrease in vigour, gross and total motility after 4 wk of scrotal insult (P < 0.001). Total defects in sperm were higher after 4 wk compared to pre-insulation and 11 wk after scrotal insulation (P < 0.001). The analysis of the 2D protein profile of the SP resulted in the identification 183 unique protein spots in all gels evaluated. There was no difference in mean number of protein spots amongst time points. Eight protein spots were more abundant in SP after scrotal insulation, returning to the same expression level at 11 wk post-insulation. One spot had higher abundance at 11 wk post-insulation, and one spot had decreased abundance 4 wk after insulation. The ten protein spots with differential abundance amongst time points were identified as Seminal plasma protein PDC-109, Seminal plasma protein A3, Seminal plasma protein BSP-30 kDa, Spermadhesin-1 and Metalloproteinase inhibitor 2. The validation of these five proteins as biomarkers for thermal testicular stress in Brangus breed would allow the development of new biotechnologies that could improve bovine semen analysis in breeding systems in tropical and subtropical conditions. A close association between the identified BSP and Spermadhesin-1 was evidenced in protein-protein interaction analysis. Based on gene ontology analysis, variation in sperm function after insulation could be explained by variation in the expressed proteins in the SP. Further studies are required to verify if these proteins could be used as biomarkers for the identification of bulls with increased seminal resistance to heat stress in Brangus breed.

Effect of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams.

The present study assessed the effects of daily supplementation with 33 mg/metabolic weight (MW) of γ-oryzanol on testicular degeneration induced by scrotal insulation in rams. Eight animals were divided into two groups: Control (subjected to scrotal insulation without treatment) and Gamma (subjected to scrotal insulation and γ-oryzanol treatment). The rams were subjected to scrotal insulation by covering the scrotum with a thermal bag for 72 h. Animals in the Gamma group received 33 mg/MW oral γ-oryzanol once-daily, beginning 7 days before insulation and continuing during insulation and for 20 days afterward, for a total treatment period of 30 days. Samples of semen and blood were collected during the experiment to perform biochemical evaluations of oxidative stress, seminal kinetics and morphology, and plasma testosterone concentrations. Ultrasound examinations of the testicular parenchyma and clinical evaluations of its consistency and the scrotal perimeter were also performed at weekly intervals. Testicular tissue was collected for biochemical analyses of oxidative stress parameters at the end of the experiment by orchiectomy. The results showed that testicular degeneration was induced by scrotal insulation, as was demonstrated by the reduced scrotal perimeter and increased in testicular flaccidity immediately after insulation. Moreover, a delayed increase in the number of hyperechoic points in the parenchyma and a delayed reduction in sperm motility were observed at 10 weeks after insulation by ultrasonography. Treatment with γ-oryzanol reduced levels of reactive oxygen species (ROS) levels in the testes, and increased the total antioxidant potential (assessed based on the ferric reducing ability (FRAP)) in week 10 and levels of lipid peroxidation (TBARS). It also increased the number of intact spermatozoa in week 3, but increased the total number of sperm defects from week 5 onwards. Although γ-oryzanol protected the semen and testes by reducing the levels of the parameters of oxidative stress evaluated herein, the other parameters studied were not improved by the treatment. In addition, supplementation with γ-oryzanol led to more morphological abnormalities in the sperm. This study presented new information on the oral administration of γ-oryzanol to rams with testicular degeneration, and described potential therapies for this pathology, which currently has no established treatment and has important impacts on reproductive health.

Effect of racial crossing on the scrotum-testicular biometry in sheep submitted to scrotal insulation / Efeito do cruzamento racial sobre a biometria escroto-testicular em ovinos submetidos à insulação escrotal

The objective of this study was to evaluate the effect of the racial crossing on scrotum-testicular biometric characteristics of four sheep Santa Ines and four crossbred (Santa Ines x Dorper) submitted to scrotal insulation, following the return of these characteristics to values previously reported. For this, two measurements were made regarding the scrotal circumference (SC), length (L) and testicular width (W) before treatments. The testicular volume (V) was calculated by the formula V=2 [(r2) x π x H]. The pouches were made with double-layer plastic, internally lined with cotton, and fixed around the spermatic funiculus and scrotum with adhesive tape and bandage remaining in this position for seven days. Measurements were made every seven days, totaling 15 measurements throughout the experiment. The data were submitted to analysis of variance (ANOVA) to a randomized block design with two blocks, 15 treatments and four replications. The variables analyzed were subjected to Dunnett test at 5% probability, to compare the values obtained before and after insulation. For comparison between the breeds, the variables were submitted to Tukey test at 5% probability. All animals studied suffered significant influence (P<0.05) after scrotal insulation, but Santa Ines returned to the values previously observed in a shorter period than the crossbred. In summary, sheep Santa Ines have a higher resistance than crossbred animals when subjected to thermal stress induced by scrotal insulation.(AU)

Objetivou-se neste trabalho avaliar o efeito do cruzamento sobre as características biométricas escroto-testiculares de carneiros da raça Santa Inês e mestiços (Santa Inês x Dorper), submetidos à insulação escrotal, acompanhando o retorno dessas características aos valores previamente observados. Para isso, foram realizadas duas mensurações referentes à circunferência escrotal (CE), comprimento (C) e largura testicular (L), antes da insulação. O volume testicular (V) foi calculado pela fórmula V=2 [(r2) x π x h]. As bolsas de insulação foram fixadas ao redor do funículo espermático e escroto com fita adesiva e esparadrapo, permanecendo por sete dias. Após o período de insulação, as mensurações foram realizadas a cada sete dias, totalizando 15 mensurações durante todo o experimento. Os dados obtidos foram submetidos à análise de variância (ANOVA) para um delineamento em blocos casualizados, com dois blocos, 15 tratamentos e quatro repetições. As variáveis analisadas foram submetidas ao teste de Dunnett a 5% de probabilidade, para comparar os valores obtidos antes e após a insulação. Para a comparação entre as raças, as variáveis foram submetidas ao teste de Tukey a 5% de probabilidade. Todos os animais estudados sofreram influência significativa (P<0,05) após a insulação escrotal, porém os carneiros da raça Santa Inês retornaram aos valores anteriormente observados em um espaço de tempo mais curto do que os mestiços. Conclui-se que carneiros da raça Santa Inês apresentam maior resistência do que animais mestiços quando submetidos ao estresse térmico induzido pela insulação escrotal.(AU)

Effect of increased testicular temperature on seminal plasma proteome of the ram.

The present study evaluated the effects of heat stress on the ram seminal plasma proteome. Six Morada Nova rams were scrotal insulated for 8 days. Scrotal circumference, sperm parameters, and seminal fluid proteins were evaluated before (Day 0) and twice during scrotal insulation (Days 4 and 8), and weekly until semen parameters returned to preinsulation values (normal). Seminal proteins were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Scrotal circumference decreased from 30 ± 0.4 cm on Day 0 to 22.6 ± 0.6 cm on Day 36 (P < 0.05) and became equivalent to preinsulation values on Day 71. Motile sperm became nearly absent from Day 8 to Day 64 but returned to normal on Day 113. Percentage of normal sperm changed similarly and returned to normal on Day 106. Rams were azoospermic between Days 29 and 64, and sperm concentration came back to normal on Day 92. The number of spots/two-dimensional gel reduced from 256 ± 31 on Day 0 to 104 ± 14 on Day 29 (when rams were azoospermic) and then increased to 183 ± 9 on Day 113 (P < 0.05), similar to spot counts before insulation. The intensities of 24 spots, referring to 17 seminal plasma proteins, were affected by treatment (P < 0.05). After insulation, seminal plasma had greater expression of actin (two isoforms), albumin, heat shock protein 70 kDa, protein DJ-1, HRPE773-like, C-reactive protein precursor, bodhesin-2 (one isoform), spermadhesins. Most protein spots had the greatest intensity between Days 8 and 29, returning to preinsulation values on Day 113 (when many sperm criteria returned to normal). Proteins downregulated after scrotal insulation included dipeptidyl peptidase 3, isoforms of heat shock protein 90 kDa, RSVP22, MMP2 and of Bdh2. In this case, RSVP22 was reduced on Day 113 and all others, on Day 134. Expression of MMP2 and HSP90.1 was reduced throughout the study. Integrin ß5, V-type H(+)-ATPase subunit A, ZBTB 42-like protein, isoforms of Bdh2, PSP-I, and RSVP22 were upregulated after testis insulation. Intensities of these spots were maximum (P < 0.05) 8 days after insulation started or on Day 29. Expression of most of such proteins returned to normal on Day 113. In conclusion, scrotal insulation affected testis and sperm parameters of rams, indicating alterations in both spermatogenesis and sperm maturation. Changes of seminal plasma proteome were coincidental with variations in semen parameters. Proteins affected by heat challenge are potentially involved in sperm protection, maturation, and fertilization.