The antioxidant effects of butylated hydroxytoluene on cryopreserved goat sperm from a proteomic perspective.

At present, there are few reports about the proteomics changes provoked by butylated hydroxytoluene (BHT) supplementation on cryopreserved semen in mammals. Thus, we aimed to evaluate the effects of different concentrations of BHT on goat sperm and to investigate the proteomics changes of adding BHT to cryopreserved goat (Capra hircus) sperm. Firstly, semen samples were collected from four goats, and frozen in the basic extenders containing different concentrations of BHT (0.5 mM, 1.0 mM, 2.0 mM) and a control without BHT, respectively. After thawing, the protective effects of dose-dependent replenished BHT to the freezing medium on post-thaw sperm motility, integrities of plasma membrane and acrosome, reactive oxygen species levels were confirmed, with 0.5 mM BHT being the best (B group) as compared to the control (without BHT, C group). Afterwards, TMT-based quantitative proteomic technique was performed to profile proteome of the goat sperm between C group and B group. Parallel reaction monitoring was used to confirm reliability of the data. Overall, 2,476 proteins were identified and quantified via this approach. Comparing the C and B groups directly (C vs. B), there were 17 differentially abundant proteins (DAPs) po-tentially associated with sperm characteristics and functions were identified, wherein three were upregulated and 14 were downregulated, respectively. GO annotation analysis demonstrated the potential involvement of the identified DAPs in metabolic process, multi-organism process, reproduction, reproductive process, and cellular process. KEGG enrichment analysis further indicated their potential roles in renin-angiotensin system and glutathione metabolism pathways. Together, this novel study clearly shows that BHT can effectively improve quality parameters and fertility potential of post-thawed goat sperm at the optimal concentration, and its cryoprotection may be realized through regulation of sperm metabolism and antioxidative capability from the perspective of sperm proteomic modification.

Beneficial effects of oral antioxidant supplementation on semen quality parameters, reproductive hormones, and sperm DNA integrity in men with idiopathic oligoasthenoteratozoospermia.

OBJECTIVE: Recently, oral antioxidants in combined forms have been used to treat men with idiopathic infertility. This study aimed to evaluate the effects of treatment with vitamin C, vitamin E, selenium, zinc, arginine, L-carnitine, and coenzyme Q10 on sperm quality parameters, DNA integrity, reproductive hormones, and pregnancy rates in men with infertility and idiopathic oligoasthenoteratozoospermia (OAT). METHODS: A prospective study was conducted on 420 men with infertility and idiopathic OAT who took an oral supplement of antioxidant SP-Power tablets twice daily for 6 months. Semen quality, reproductive hormones, and the DNA fragmentation index (DFI) were evaluated at baseline and at 3 and 6 months after supplementation, using the World Health Organization 2021 guidelines. RESULTS: No significant difference was observed in volume or the percentage of typical morphology during treatment. A significant improvement in sperm concentration was observed after supplementation (8.67±1.41, 12.17±1.91, and 19.01±0.86 at baseline, 3, and 6 months respectively, p<0.01). The total motility, progressive motility, and total motile sperm count also increased significantly (p<0.01), whereas the DFI decreased after 6 months. There was an increase in normal FSH levels and testosterone levels after 6 months of supplementation of antioxidant SP-Power but these differences were not statistically significant (p=not significant and p=0.06, respectively). CONCLUSION: Supplementation with SP-Power tablets improved sperm quality parameters, sperm DFI, some reproductive hormones, and pregnancy rates in men with infertility and idiopathic OAT, which could be attributed to the supplement's synergistic antioxidant action. Further studies are needed to determine the effects of supplementation on oxidative stress markers.

The Addition of Opuntia ficus-indica Ethanolic Extract to a Skimmed Milk-Based Extender Impacts Ram Sperm Quality.

Recently, researchers have focused on the use of natural antioxidants to improve semen quality as a key element for successful artificial insemination. In this context, the first aim of this study was to determine the antioxidant activity and composition (minerals, vitamins, and sugars) of Opuntia ficus-indica cladode ethanolic extract (ETHEX). A further purpose of the study was to investigate the effect of ETHEX supplementation on the quality of liquid ram semen extended with skim milk (SM) at 5°C. The antioxidant activity of ETHEX was studied using free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH•) assay. The mineral composition and the sugar and vitamin contents of ETHEX were determined using an inductively coupled plasma optical emission spectrometry (ICP-OES) and HPLC-DAD-RID analytical instruments. As a second part, semen was collected from five Boujaâd rams with an artificial vagina. The ejaculates with more than 70% motility were pooled, extended with skim milk (SM) extender without (control) or supplemented with 1-8% of ETHEX (37°C; 0.8 × 109 sperm/mL). Sperm quality parameters were assessed at 8, 24, 48, and 72 h. The results showed that ETHEX had a higher antioxidant activity compared to those of ascorbic acid and butylated hydroxytoluene (BHT). Furthermore, ETHEX contains a considerable amount of minerals, vitamins, and sugars. The inclusion of 1 or 2% ETHEX in SM increased the sperm motility, viability, and membrane integrity and decreased the abnormality of spontaneous and catalyzed lipids peroxidation (p < 0.05) up to 72 h. In addition, semen diluted with 1 and 2% ETHEX decreased the level of DNA fragmentation compared to the control group (p < 0.05). In conclusion, the ETHEX could be recommended to improve the quality of liquid ram spermatozoa. However, its effects on artificial insemination should be further studied.

Does advanced age affect reproductive variables, semen composition, and liquid semen storage during different seasons in Boujaâd rams?

The present research was conducted to evaluate the effects of season and age on scrotal circumference (SC), semen characteristics, semen composition, and sperm motility during liquid storage in Boujaâd rams. Semen was collected throughout 1 year from April 2014 to March 2015. Two ram groups, namely older rams including animals aged 5.5 to 6 years of age, and younger rams including animals aged 2.5 to 3 years of age were used. Scrotal circumferences, semen characteristics, and some biochemical variables from semen were measured. After collection and evaluation, the semen was extended in skim milk (SM) at 15 °C to a concentration of 0.8 × 109 spermatozoa/ml. Thereafter, samples were evaluated at different storage times (0, 8, and 24 h). The results indicate that older rams had greater values of SC, lesser semen quality, protein, lipid, and cholesterol concentrations than younger rams. Nevertheless, for both groups there were the least values for semen quality and semen composition in winter. The semen collected from younger rams had greater motility after liquid storage compared with that of the older rams. The least semen quality after liquid storage was recorded in the winter compared with the other seasons of the year for both age groups. In conclusion, results of the present study indicate that older Boujaâd sheep have lesser semen quality, different semen composition than younger rams after liquid storage and during different seasons of the year. Furthermore, there was a strong correlation between semen composition and motility of stored ram semen.

Morphometry and depth of inseminating catheter penetration in prolific and non- prolific ewes at different ages: A post mortem study.

The ewe's cervix structure is highly variable among females, which may limit the application of transcervical artificial insemination (TCAI). This work aimed to study the variation in cervix anatomy in two different prolificacy breeds (D'man a prolific and Boujaâd a non-prolific sheep). Reproductive tracts were collected from D'man (n = 64) and Boujaâd (n = 187) females aged between 1 and 4 years. The weight of the uterus, length of the cervix and depth of penetration of the inseminating pipette were recorded. The number of cervical rings was counted and their arrangement graded. The cervix anatomy of D'man ewes differs from that of Boujaâd. The cervix morphology of Boujaâd ewes is more complex compared to D'man ewes; this complexity depends on the age of the ewe. No significant difference was found regarding the depth of penetration of the insemination catheter for the two breeds at 1, 2 and 4 years old (P > 0.05). However, at 3 years old, the highest depth of penetration (P < 0.05) was recorded in D'man ewes. In addition, for both breeds, grade 2 was the most frequent (P < 0.05), and the cervix became less complicated for TCAI with advanced age (4 years old). To conclude, the cervix of D'man ewes is less complex and more favorable to TCAI. In addition, to overcome the complexity of the cervix in Boujaâd sheep, a selection of 4-year-old ewes will make the penetration of the artificial insemination catheter possible.

Supplementation of ram semen extender to improve seminal quality and fertility rate.

In sheep, artificial insemination serves as an important technique for breed improvement. In this context, genetic material from a small number of superior sires can be used in a large number of females. During this process, the storage of ram sperm may influence the efficiency of artificial insemination. Two main methods are currently used for ram semen storage: liquid storage and cryopreservation. The oxidative stress during the storage process can injure ram sperm and in some cases this leads to irreversible damage at the cellular level. To reduce such negative effects, different preservation protocols, extenders and protective components have been tested to improve ram sperm quality and to achieve greater fertility rates. This review provides an overview of the recent progress in extender supplementation using antioxidants and other compounds to improve ram semen quality parameters and fertility rates. It will emphasize on enzymes, vitamins, amino acids, proteins, some plant extracts and other compounds such as sugars, seminal plasma and fatty acids that can be used to supplement the extenders to reduce the formation of oxidants in ram semen and maintain its quality and enhance its fertility. It will also stress on how these supplements act, what were the tested levels giving beneficial effects on motility, viability, plasma membrane integrity and DNA fragmentation in liquid, cooled and post-thawing semen?

Could a bovine pregnancy rapid test be an alternative to a commercial pregnancy-associated glycoprotein ELISA test in dairy cattle?

The aim of the present study was to compare a Bovine Pregnancy Rapid Test (Ubio quickVET; BPRT) with a commercial ELISA-PAG test (Bovine Pregnancy Test DG29®) for early pregnancy diagnosis based on the presence of the PAG (pregnancy-associated glycoprotein) in dairy cattle between 30 and 40 days after artificial insemination (AI). Blood samples were collected from 212 cows between 30 and 40 days after artificial insemination (AI) to quantify the concentrations of PAG in each sample. Transrectal ultrasonography (TRUS) diagnosis of pregnancy was conducted at day 45 ±â€¯3 after AI as the reference standard for the two PAG tests. The results indicated the sensitivity (Se) of the BPRT and DG29 for diagnosing pregnant cattle were 89.4% and 100%, respectively while the specificity (Sp) of the two tests for diagnosis of non-pregnant animals was 89.8% and 81.3%, respectively. Based on these results, the BPRT has a slightly lesser sensitivity and greater specificity than the DG29 test. Moreover, the accuracy of both tests was 94% and 90% respectively for DG29 and BPRT. This implies that the BPRT test could be considered an accurate test when compared to PAG-ELISA test and TRUS at days 30-40 after AI. The BPRT test, therefore, can be used as an alternative to the PAG-ELISA test with some constraints that need to be considered with its use.

Effect of season on scrotal circumference, semen characteristics, seminal plasma composition and spermatozoa motility during liquid storage in INRA180 rams.

The present study was undertaken to assess the effect of seasons on scrotal circumference, semen characteristics, seminal plasma composition, and sperm motility during liquid storage of INRA180 rams. The semen was collected from five mature INRA180 rams (2-3 years of age) during one year (from April 2014 to March 2015). Scrotal circumferences, semen characteristics, some biochemical parameters of seminal plasma were evaluated. Immediately after collection and evaluation, the semen was pooled and extended in skim milk (SM) at 15°C to reach 0.8×109 spermatozoa/ml. Thereafter, samples were evaluated at different storage times (0, 8, and 24h). The results showed that scrotal circumference, semen quality and the concentration of total protein in seminal plasma were relatively constant during the year (P>0.05). However, total lipid and cholesterol concentrations increased significantly (P<0.001) in winter and summer. The result showed also that progressive motility was higher in winter and summer after 24h of storage (P<0.01). In contrast, no difference was recorded regarding total motility (P>0.05). To conclude, the INRA180 rams have the ability to produce semen with high quality all over the year. The only parameters showing seasonal variations are cholesterol, total lipid, and progressive motility.

Protective effects of Opuntia ficus-indica extract on ram sperm quality, lipid peroxidation and DNA fragmentation during liquid storage.

The present study aimed to assess the phenolic composition of the acetone extract from Opuntia ficus indica cladodes (ACTEX) and its effects on ram semen variables, lipid peroxidation and DNA fragmentation during liquid storage at 5°C for up to 72h in skim milk and Tris egg yolk extenders. Semen samples from five rams were pooled extended with Tris-egg yolk (TEY) or skim milk (SM) extenders containing ACTEX (0%, 1%, 2%, 4% and 8%) at a final concentration of 0.8×109 sperm/ml and stored for up to 72h at 5°C. The sperm variables were evaluated at different time periods (8, 24, 48 and 72h). Sperm total motility and viability were superior in TEY than in SM whereas the progressive motility, membrane integrity, abnormality and spontaneous lipid peroxidation were greater in SM compared to TEY (P<0.05). The results also indicated that the inclusion of 1% ACTEX in the SM or TEY extender increased the sperm motility, viability, membrane integrity, and decreased the abnormality, lipids peroxidation up to 72h in storage compared to control group. Similarly, even at 72h of storage, 1% ACTEX can efficiently decrease the negative effects of liquid storage on sperm DNA fragmentation (P<0.05). In conclusion, SM and TEY supplemented with 1% of ACTEX can improve the quality of ram semen. Further studies are required to identify the active components in ACTEX involved in its effect on ram sperm preservation.

Effect of argan oil on liquid storage of ram semen in Tris or skim milk based extenders.

Due to its high antioxidant content, the argan oil could play a beneficial role in liquid storage of ram semen. The aim of this study was to investigate effects of different concentration of argan oil (ARO) on spermatologic parameters, lipid peroxidation and DNA fragmentation during liquid storage of ram semen until 48 h. Also effects of extenders and temperature on same parameters were assessed. For these aims, semen samples were collected from Boujaâd rams, extended with Tris egg yolk or skim milk extenders without (control) or supplemented with different concentrations of ARO (1%, 2%, 5% and 10% v/v) at a final concentration of 0.8 × 10(9) sperm/mL and stored until 48 h at 5 °C or 15 °C. The sperm quality assessments were performed at different intervals during storage (0, 8, 24 and 48 h). Sperm progressive motility started to decrease after 8h of storage in all temperatures--extenders combinations and dropped steadily during the 8-48 h interval. However, sperm viability, progressive motility and membrane integrity were markedly higher in ARO groups (especially in 1% in Tris and 5% in skim milk) until 24h and 48 h storage at both temperatures compared to controls. The argan oil also decreased the level of spontaneous and induced malondialdehyde (MDA) and the sperm DNA fragmentation until 48 h storage. In conclusion, it was determined that addition of argan oil to conventional extenders may improve the quality of ram semen during liquid storage in different temperatures.