Enhancing cryopreserved ram sperm quality at -80 °C with Spirulina platensis and Salvia verbenaca extracts.

This study was conducted in two steps to evaluate the influence of freezing methods and natural extracts on cryopreserved ram sperm quality. Initially, the research compared the effects of two freezing methods: liquid nitrogen (LN2) versus -80 °C, on post-thawed ram semen on total and progressive motilities and velocity parameters. Experiment I revealed no significant differences (P > 0.05) between the LN2 and -80 °C freezing methods, indicating similar effects on the analyzed parameters. Experiment II aimed to examine the influence of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to egg yolk extender on cryopreserved sperm quality, utilizing the -80 °C freezing method. Various concentrations (1.25, 3.75, 6.25 and 8.75 µg*mL-1) of acetone (Ac-SP and Ac-SV) and hexanoic (Hex-SP), as well as methanolic (MeOH-SV) extracts, were added into the extender. A thorough assessment of post-thawed sperm quality parameters, encompassing motility, velocity parameters, viability, membrane integrity, abnormality and lipid peroxidation was conducted. The outcomes demonstrated that 1.25 and 3.75 g*mL-1 of Ac-SP and Hex-SP and 1.25 µg*mL-1 of AC-SV and MeOH-SV increased the post-thawed ram sperm quality. In conclusion, this study emphasizes the antioxidant properties of SP and SV extracts, highlighting their potential to protect cryopreserved sperm cells from oxidative stress at -80 °C.

Rearing Management and Its Impact on Caseous Lymphadenitis in Sheep.

Two surveys were conducted to assess the prevalence and risk factors of ovine caseous lymphadenitis (CL) and explore the association between its superficial and visceral forms in Sardi sheep in Settat province, Morocco. A total of 1521 sheep, including 318 lambs < 6 months, 572 young sheep aged 6-24 months, and 631 adult sheep > 24 months, were examined from 57 flocks. Superficial CL prevalence levels were as follows: 69/318 in lambs, 292/572 in young sheep, and 114/631 in adult sheep. Additionally, 2124 sheep, including 1813 young and 311 adult sheep, were inspected in slaughterhouses ante- and post-mortem. Among all infected animals, the prevalence of visceral abscesses was notably higher in adult sheep (83%, OR = 9.8, 95% CI = 5.5-17.2) compared to young sheep (35%). Data on flock size, sex, age, body condition score, rearing methods, and hygienic measures were collected. Suspected CL cases were confirmed using quadruplex-PCR. Poor barn ventilation, lack of abscess control, and younger age were identified as key risk factors for CL. Additionally, a high percentage (89%) of animals with thoracic abscesses did not display superficial lesions. While age and abscess control have been reported in previous studies, barn ventilation appears to be a new, but important, risk factor. In conclusion, the identified risk factors in Settat province are linked to breeding management practices. Implementing regular abscess control measures and improving barn ventilation are essential for CL prevention.

Inclusion of Spirulina platensis and Salvia verbenaca extracts to boost semen quality and fertilization ability in sheep.

The aim of this study was to investigate the effect of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to skimmed milk (SM) extender on ram sperm quality and fertility. Semen was collected using an artificial vagina, extended in SM to reach a final concentration of 0.8 × 109 spermatozoa/mL, stored at 4°C and evaluated at 0, 5 H and 24 H. The experiment has been performed in three steps. Firstly, from four extracts (methanol: MeOH, acetone: Ac, ethyl acetate: EtOAc and hexane: Hex) of SP and SV, only acetonic and hexanoic extracts of SP and acetonic and methanolic extracts of SV showed the highest in vitro antioxidant activities and were then selected for the following step. Thereafter, the effects of four concentrations (1.25, 3.75, 6.25, and 8.75 µg/mL) of each selected extract on stored sperm motility were evaluated. The output of this trial led to select the best concentrations having beneficial effects on sperm quality parameters (viability, abnormalities, membrane integrity, and lipid peroxidation) and fertility after insemination. The results showed that the same concentration (1.25 µg/mL) of both Ac-SP and Hex-SP, as well as 3.75 µg/mL of Ac-SV and 6.25 µg/mL of MeOH-SV, maintain all sperm quality parameters at 4°C during 24 H of storage. Besides, no difference was found in fertility between the selected extracts and the control. In conclusion, SP and SV extracts were shown to improve the quality of ram sperm and to maintain fertility rate after insemination as similar or competitive to many previous studies published in the field.

Effects of Origanum majorana essential oil and antibiotics on the quality of frozen thawed Beni Arouss buck semen.

This study aims to investigate the effects of Origanum majorana (OM) essential oil (EO) at different concentrations and antibiotics on post-thawed Beni Arouss buck semen quality. Semen collection was performed using artificial vagina from eight Beni Arouss bucks. Ejaculates were pooled, divided into 12 equal aliquots, washed and diluted to 400 × 106 sperm/ml (with 7% of glycerol). Skim milk-based extender was supplemented with different concentrations of OM EO (0%; 0.01%; 0.02%; 0.03%; 0.04% and 0.05%) without antibiotics, marked as (CTR-), (M1-), (M2-), (M3-), (M4-) and (M5-), and with 50 mg of streptomycin and 50,000 IU of penicillin per 100 ml, marked as (CTR+), (M1+), (M2+), (M3+), (M4+) and (M5+), respectively. Aliquots were cooled to 4°C, then frozen in 0.25 ml straws with a programmable freezer and finally stored in liquid nitrogen for 48 h. Thawing was performed at 37°C for 30 s. Motility, live sperm, sperm abnormalities, membrane integrity, lipid peroxidation and bacterial growth were evaluated after thawing. Among the tested extenders, M2+ improved all semen quality parameters. Sperm motility, live sperm and membrane integrity increased significantly, while the number of abnormal sperm and bacterial growth decreased significantly. The toxic effect of OM EO, with and without antibiotics, appeared beyond 0.03%. In conclusion, M2+ is recommended to improve the cryopreservation of Beni Arouss buck semen.

Analysis of the Genetic Diversity and Population Structure of Four Senegalese Sheep Breeds Using Medium-Density Single-Nucleotide Polymorphisms.

In Senegal, sheep breeds have adapted to their environment and play a key socio-economic role. This study aimed to explore the genetic diversity and structure of four Senegalese sheep breeds (Peul-peul, Djallonke, Touabire, and Ladoum) and their relationships with global sheep breeds. To that end, forty-seven sheep were genotyped using the OvineSNP50 BeadChip, and these genotypic data were analysed with those of 73 sheep breeds representative of worldwide ovine diversity (2729 animals). The average observed heterozygosity (Ho) ranged from 0.293 in Djallonke sheep to 0.339 in Touabire sheep. The estimated Fis values were low, ranging from 0.019 for Ladoum to 0.034 for Peul-peul sheep. The estimated Fst values were low (0.003-0.044) among the trypanosusceptible breeds (Peul-peul, Touabire, and Ladoum) but high between the previous breeds and the trypanotolerant Djallonke breed (0.075-0.116), indicating better genetic conservation of the Djallonke sheep. A principal component analysis revealed clustering of the Senegalese sheep breeds according to their geographic distribution. However, owing to genetic improvement practices, the introgression of Touabire sheep blood seems to have reshaped the genetic landscape of the trypanosusceptible sheep breeds in Senegal. The Senegalese sheep breeds showed lower genetic diversity than their presumed ancestral sheep breeds of the Middle East. They also presented some relatedness with Caribbean sheep breeds, which reveals their contribution to the global genetic diversity and to the development of Caribbean sheep breeds.

Control of cystic echinococcosis in the Middle Atlas, Morocco: Field evaluation of the EG95 vaccine in sheep and cesticide treatment in dogs.

BACKGROUND: Cystic echinococcosis (CE) is an important cause of human morbidity and mortality worldwide, particularly in Morocco and other North African countries. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the potential of three strategies to reduce Echinococcus granulosus transmission: (1) 4-monthly treatment of dogs with praziquantel, (2) vaccination of sheep with the EG95 vaccine and (3) a combination of both measures. These measures were implemented during four consecutive years in different areas of the Middle Atlas Mountains in Morocco. The outcome of the interventions was assessed through hydatid cyst (viable and non-viable) counts in liver and lungs using necropsy or in vivo ultrasound examination of the liver. A total of 402 lambs were recruited for annual vaccination with the EG95 anti-E. granulosus vaccine and 395 similar lambs were selected as non-vaccinated controls. At approximately four years of age the relative risk (estimated as odds ratio) for vaccinated sheep to have viable hydatid cysts compared with non-vaccinated controls was 3% (9.37% of the vaccinated sheep were found infected while 72.82% of the controls were infected; p = 0.002). The number of viable cysts in vaccinated animals was reduced by approximately 97% (mean counts were 0.28 and 9.18 respectively; p<0.001). An average of 595 owned dogs received 4-monthly treatment during the 44 months trial, corresponding to 91% of the owned dog population. Approximately, 5% of them were examined for E. granulosus adult worms by arecoline purge or eggs in feces (confirmed by PCR). The proportion of infected dogs significantly decreased after treatment (12% versus 35%; p<0.001). Post-treatment incidence of re-infestation corresponded to a monthly risk of 4% (95% CI: 3-6%). Treatment of owned dogs on a 4-monthly basis did not reduce the level of transmission of E. granulosus to sheep, nor did it enhance the level of control generated by vaccination of sheep with EG95, possibly because of unowned dogs and wild canids were not treated. CONCLUSIONS/SIGNIFICANCE: These data suggest that vaccination of sheep with EG95 has the potential to reduce the level of CE in Morocco and in other parts of the world with similar transmission dynamics. Under the epidemiological circumstances existing in the trial area, 4-monthly treatment of owned dogs with praziquantel was insufficient to have a major impact of E. granulosus transmission to sheep.

Influence of Season and Liquid Storage at 16 °C on Beni Arouss Bucks' Semen Quality.

The study aimed at determining the effect of storage and season on fresh semen of Beni Arouss goats. Ejaculates were collected at monthly intervals from seven mature bucks and were extended at a final concentration of 800 × 106 spermatozoa. ml-1 and stored at 16 °C for 24 h. Semen motility, viability and normal morphology were assessed at 0, 4, 8 and 24 h after collection. Motility and normal morphology parameters were recorded using computer-assisted sperm analysis (CASA) and viability was analyzed using eosin-nigrosin staining. As expected, motility, viability and normal morphology parameters showed a significant reduction within 24 h of storage and during all seasons (p < 0.05). However, semen collected in summer maintained a better quality after 24 h of storage at 16 °C than semen collected during the other periods (p < 0.05). In conclusion, the storage ability of Beni Arouss bucks' semen stored at 16 °C was significantly higher during the summer.

Onset of puberty and regularity of oestral cycles in ewe lambs of four breeds under high-altitude conditions in a non-seasonal country.

Twenty-four Colombian Creole, Romney Marsh, Hampshire and Corriedale ewes raised under high-altitude conditions in a non-seasonal country were used to determine and to characterize the onset of puberty and of regularity ovarian function since the age of 3months. They underwent blood collection for determination of progesterone and monthly weight assessment. Oestrus was assessed daily by a teaser ram. In all breeds, age at onset of puberty ranged from 5.4 to 6.9 months and age at onset of regular ovarian function ranged from 7.4 to 8.6 months. Colombian Creole showed a higher body development at onset of puberty: 73.5 ± 8.3% versus 56.2 ± 7.4 in Romney Marsh, 58.8 ± 10.4 in Hampshire, and 57.3 ± 8.0 in Corriedale (p < 0.05), as well as a higher relative daily weight gain (+17%, p < 0.05). A negative correlation between daily weight gain and age at onset of puberty was established. Progesterone at onset of puberty and of regularity did not differ between breeds, but characterization of the luteal phase by the progesterone area under the curve (P4-AUC) revealed at both time points significantly lower values for Creole lambs (p < 0.05). Decreased P4-AUC paralleled a higher proportion of short oestral cycles observed prior to onset of regularity in Creoles, whereas an increased proportion of extra-long cycles were recorded in Romney Marsh (p < 0.05). These results establish first reference data for economically important breeds raised under tropical conditions. In comparison with other breeds, Colombian Creole requires a higher body development to achieve puberty and that an important proportion of short characterizes its prepubertal period cycles.

The socio-economic burden of cystic echinococcosis in Morocco: A combination of estimation method.

Cystic echinococcosis (CE) is a major zoonosis in Morocco despite the launch of a national control programme in 2005. As its economic consequences have not been studied yet in Morocco, this study estimated CE impact in terms of monetary losses, disability-adjusted life years (DALY), and DALY for zoonotic diseases (zDALY) in the entire country and in specific regions for the 2011 to 2014 period. The direct monetary losses were related to organ seizure from infected animal in slaughterhouses, and to healthcare expenses as well as lost wages for infected humans. Animal production losses concerned milk yield, fertility, carcass weight, and wool production. Losses due to human infection were also composed of disability and productivity losses at work. Monte Carlo simulations were used to estimate monetary losses and zDALY values. Nationwide, the estimated DALY was 0.5 years per 100,000 persons per year, and the zDALY was 55 years per 100,000 persons per year. Total yearly losses were estimated at 73 million USD (54-92 million USD). However, losses differed significantly among regions. Most of the economic losses consisted of unperceived consequences, i.e. decreased animal production and reduced productivity of asymptomatic individuals. Future studies should determine the socioeconomic and epidemiological factors underlying the differences in economic losses among regions to develop better adapted control programmes.

Evaluation of different hormonal treatments on oestral and ovarian responses in Moroccan Beni Arouss goats during anoestrus and breeding season.

The efficacy of eight combinations of fluorogestone acetate (FGA, 20 or 40 mg as intravaginal device during 11 days), equine chorionic gonadotropin (eCG, 300 or 500 UI injected 48 hr before FGA removal) and prostaglandin F2α (cloprostenol, 0 or 50 µg injected 48 hr before FGA removal) aiming at induction and synchronization of oestrus and ovulation was evaluated during the anoestrus season in spring and during the breeding season in autumn in adult Beni Arouss goats. Oestrous behaviour was recorded between 12 and 60 hr after FGA removal. Blood samplings allowing to assess onset of the pre-ovulatory LH surge and increase of progesterone as sign of an active corpus luteum were performed, respectively, between 20 and 60 hr and 3, 5, 8 and 15 days after FGA removal. No season-related differences (spring vs. autumn) were observed for oestrous response (95% vs. 93%), pre-ovulatory LH surge (94% vs. 84%) and luteal response after 3-8 and 11-15 days post-treatment (respectively 92% vs. 66% and 92% vs. 98%). The onset of oestrus (21 [13-53] vs. 32 [12-54] hr) and LH surge (26 [20-60] vs. 38 [22-60] hr) occurred significantly later in autumn. FGA (40 vs. 20 mg) in autumn significantly delayed the onset of oestrus (36 [16-54] vs. 23 [12-47] hr) and LH surge (44 [26-58] vs. 33 [22-60] hr). Significant treatment-related differences were recorded for onset of LH surge (earliest for 20 mg FGA, 300 IU eCG, 50 µg PGF2α ) and onset of luteal phase (latest for 40 mg FGA, 300 IU eCG, 50 µg PGF2α ). In conclusion, the hormone combinations tested appeared equally effective in terms of oestrous and ovulation rates. Season has influenced significantly the onset of oestrus and LH surge, and the high dose regimen of FGA delayed the ovarian response in autumn.

Effect of season on sexual behavior, testicular measurements and seminal characteristics in "Beni arouss" North Moroccan bucks.

Changes in anatomical, behavioural and seminal characteristics were measured throughout the year in seven Beni Arouss bucks maintained at a latitude of 35 °N. Testicular size varied significantly. There were the greatest values during the summer and autumn for scrotal circumference and least in the winter for testicular length. Values for most variables related to sexual behavior were not affected by season except the number of mounts before first ejaculation which was least in the summer. No differences were detected between seasons in terms of semen volume. The sperm concentration, viability and percentage of normal sperm, however, were less during the winter period. The size of sperm heads was larger in the autumn and motility of spermatozoa was greater in the summer and autumn. Results from the correlation analysis indicated that the majority of conventional seminal characteristics were correlated with testis measurements (P < 0.05; r < 0.5). Values for conventional and CASA motility variables indicated there were positive correlations between viability and progressive motility (r = 0.4; P < 0.05) and between the percentage of normal sperm and straight line and average path velocity (r = 0.4; P < 0.05). A global reproduction performance score was established for each buck, which allowed for assessment of magnitude of seasonal changes for each individual. These global score values indicated there was a greater reproductive performance for all bucks during summer and autumn. This study described for the first time seasonal variations of reproductive characteristics of Beni Arouss bucks and results indicate that even though there is a maximal capacity for reproductive performance during the summer and autumn, breeding should be possible throughout the year.

Effect of an inactivated bluetongue serotype 8 vaccine on semen quality in rams.

The aim of this study was to determine whether a single dose of an inactivated bluetongue virus serotype 8 (BTV-8) vaccine altered semen quality in rams. Twenty sexually mature rams were assigned to three experimental groups: two groups of four animals were vaccinated and a third group of four animals was unvaccinated. The first group included rams with a history of natural BTV-8 infection in 2007 and the second and third groups included BTV-8 naïve rams. Semen was collected prior to vaccination and for 4 months post-vaccination. There were no significant differences in semen quality traits, including motility and concentration of spermatozoa, and percentages of living, normal dead and abnormal dead spermatozoa, between vaccinated and unvaccinated groups, or over time (P>0.05). The BTV-8 vaccine tested in this study did not appear to have any adverse effect on semen quality in rams.

Viral RNA load in semen from bluetongue serotype 8-infected rams: relationship with sperm quality.

This study investigated if viral RNA was detectable in the semen of rams clinically infected with bluetongue virus serotype 8 (BTV-8) by RT-qPCR, and to what extent the amount detected may be predictive of sperm quality. Semen samples were collected on six occasions from 93 BTV-8 infected rams involved in two longitudinal (n=12 and 27, respectively) and one cross-sectional (n=54) field study. Semen quality was assessed in terms of mass motility, concentration of spermatozoa, percentage of living and dead spermatozoa as well as cytological features. An overall semen quality score (SQS) was established. Depending upon the studied population, BTV RNA was detected in 75-100% of semen samples at initial testing 25-57 days post-observation (DPO) of clinical signs, and was detectable up to 116 DPO in a proportion of rams undergoing repeated sampling. Semen quality variables were significantly altered following natural BTV-8 infection and correlated with the amount of BTV RNA present. The SQS did not return to normal when virus was no longer detectable, suggesting that clearance of BTV precedes full recovery of sperm quality. In conclusion, viral RNA may be transiently recovered from the semen of BTV-8 affected rams and may serve as an indicator in predicting ram breeding potential following natural infection.

The impact of naturally-occurring, trans-placental bluetongue virus serotype-8 infection on reproductive performance in sheep.

Infection with bluetongue virus serotype (BTV)-8 occurred in ruminants in 2006 in Central-Western Europe. The trans-placental passage of this virus has been demonstrated in naturally- and experimentally-infected cattle and in experimentally-infected sheep. Trans-placental transmission is potentially important in the 'over-wintering' of this virus and its subsequent impact on reproductive performance. This epidemiological study was carried out on a sheep flock in Belgium that had experienced a severe outbreak of BTV-8 infection, and where the seroprevalence had increased from 1.3% to 88% between January and November 2007. In total, 476 lambs and 26 aborted fetuses from 300 ewes, lambing at four distinct time periods, were investigated between November 2007 and May 2008. The following evidence suggested that BTV-8 infection occurred in utero: (1) positive PCR results from splenic tissue from aborted fetuses (n=4); (2) fetal malformations suggestive of BTV infection (n=10); (3) positive PCR results from red blood cells in-lambs (n=7), and (4) the presence of antibody at birth in viable lambs prior to the intake of colostrum (n=9). The evidence provided by this investigation strongly suggests that trans-placental BTV-8 infection occurs in naturally-infected sheep and the impact of infection on the reproductive performance of such a naïve flock was considerable, with up to 25% of ewes aborting and with flock fertility reduced by 50%. The contribution of in utero-infected lambs to the over-wintering of BTV appears limited.

Impact of a natural bluetongue serotype 8 infection on semen quality of Belgian rams in 2007.

In 2006, bluetongue (BT) virus serotype 8 emerged in northern Europe and numerous ruminants were affected in the following year. Infertility in males is one of the consequences of BT, although its severity and duration after natural infection has not been documented. In this report, the impact of BT-8 on clinical signs and semen quality of naturally infected rams is described through a longitudinal study of two Belgian ram populations (n=12 and n=24) and a cross sectional study in a further ram population (n=43). Macroscopic semen characteristics, semen concentration, motility, percentage of living and dead spermatozoa were assessed in 167 semen samples collected on 1-6 occasions from 79 BT-8 infected rams within 5-138 days after onset of clinical disease. These were compared with healthy control animals. Significant changes in all variables were observed after natural BT-8 infection. Total recovery occurred around 85 days after clinical disease in animals undergoing a close follow-up of semen quality. Good correspondence between the results of the longitudinal and cross sectional studies suggests that semen quality of BT-8 affected rams reached normal references values 63-138 days after clinical diagnosis of BT. In addition, semen concentration seems to be a sound epidemiological indicator of ram semen quality.