Valentina Longobardi - Academia.edu (original) (raw)
Papers by Valentina Longobardi
Animal Reproduction Science
Scientific Reports, Sep 1, 2022
In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one ... more In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one of the major factors affecting farming profitability. Follicular fluid (FF) contains extracellular vesicles (EVs) playing an important role in modulating oocyte developmental competence and carrying microRNAs (miRNAs) essential for in vitro fertilization outcomes. The aim of this work was to characterize the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) follicles collected in the breeding (BS) and non-breeding (NBS) seasons, to unravel the molecular causes of the reduced oocyte competence recorded in buffalo during the NBS. In total, 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) were found. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR < 0.05) between An and pO groups. A subset of the most significant DE-miRNAs between An and pO groups targets genes which function is related to the lipid and steroid metabolism, response to glucocorticoid and oestradiol stimulus. Comparison between BS and NBS showed 14 and 12 DE-miRNAs in An-FF-EVs and pO-FF-EVs, which regulate IL6 release and cellular adhesion, respectively. In conclusion, these results demonstrated that the miRNA cargo of buffalo FF-EVs varies in relation to both follicular development and season. The importance of buffalo (Bubalus bubalis) breeding is clearly indicated by the positive growth trend all over the world (Faostat.fao.org/faostat), due to specific features making this species a valuable protein source, particularly for tropical countries. A peculiar situation is described in Italy, where the success of buffalo breeding is closely related to the production of mozzarella cheese, highly requested around the world. A major limiting factor is reproductive seasonality, impeding continuity of milk production throughout the year, and hence not allowing to meet the market demand. Buffalo is a short-day breeder, with an increased reproductive activity observed during decreasing day length months 1,2. In Italy, where the Italian Mediterranean buffalo breed has been selected, the seasonality pattern shows an opposite trend to the market request. Therefore, the out of breeding mating strategy (OBMS), has been efficiently applied to distribute calving more evenly during the year 2. However, forcing buffalo cows to conceive during the non-breeding season (NBS) may lead to extended post-partum anestrus, higher rates of embryonic mortality and overall reduced fertility 1,3,4. In previous studies it was demonstrated that embryonic mortality is in part caused by impaired luteal function, and consequently reduced progesterone secretion 5. This in turn interferes with embryo growth that is accompanied by transcriptomic and proteomic changes at the level of embryos and chorioamnios/caruncles 6,7 , definitively hampering embryo attachment. Embryonic mortality during the NBS is also in part due to reduced oocyte developmental competence, as shown by the decreased cleavage and blastocyst rates obtained after in vitro fertilization during increasing daylight months in Italian Mediterranean buffaloes 8,9. A seasonal effect on follicular population and embryo outcomes was also reported by other authors 10,11. A poorer oocyte quality was observed in Murrah buffalo heifers during
Theriogenology, 2017
The aim of this study was to evaluate the effect of carnitine supplementation of semen extender o... more The aim of this study was to evaluate the effect of carnitine supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. Buffalo semen was cryopreserved in BioXcell containing 0 (control group), 2.5 and 7.5-mM carnitine. After thawing, viability, motility, membrane integrity and capacitation status (assessed by localization of phosphotyrosine-containing proteins and chlortetracycline, chlortetracycline assay) were evaluated. Furthermore, total antioxidant capacity, reactive oxygen species (ROS) and lipid peroxidation levels, as well as adenosine triphosphate (ATP) content and phospholipids concentration were assessed. Finally, in vitro-fertilizing ability was evaluated after heterologous IVF. An increased post-thawing sperm motility and membrane integrity were recorded in both treated groups compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6%; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.05 and 48.44 ± 0.69, 55.19 ± 0.54, 59.63 ± 0.30%; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01 with 0, 2.5-mM, and 7.5-mM carnitine, respectively). Supplementation of carnitine to the freezing extender decreased (P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01) the percentage of sperm displaying fluorescence at both equatorial and anterior acrosomal regions (pattern EA), corresponding to high capacitation level, compared with the control (30.3 ± 3.8, 18.8 ± 2.8, and 7.2 ± 2.9%, respectively, with 0, 2.5-mM, and 7.5-mM carnitine). In agreement with this, carnitine also decreased (P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01) the percentage of sperm displaying chlortetracycline pattern B (capacitated sperm) (63.8 ± 1.8, 46.8 ± 2.2, and 37.2 ± 1.8%, respectively with 0, 2.5-, and 7.5-mM carnitine). Interestingly, carnitine increased total antioxidant capacity and ATP content of buffalo frozen-thawed sperm (1.32 ± 0.02, 1.34 ± 0.01, 1.37 ± 0.01 mM/L and 4.1 ± 0.1, 5.3 ± 0.1 and 8.2 ± 0.4 nM × 10(8) sperm; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01, respectively, with 0, 2.5- and 7.5-mM carnitine). Intracellular ROS decreased in carnitine-treated sperm compared with the control, as indicated by dihydroethidium (DHE) values (0.22 ± 0.01, 0.18 ± 0.01, and 0.14 ± 0.0 μM/100 μL dihydroethidium, respectively, with 0, 2.5-, and 7.5-mM carnitine; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01), whereas lipid peroxidation levels (on average 30.5 ± 0.3 nmol/mL MDA) and phospholipids concentration (on average 0.14 ± 0.00 μg/120 × 10(6) sperm) were unaffected. Despite the improved sperm quality, the percentage of normospermic penetration after IVF was not influenced (on average 53.5 ± 1.8). In conclusion, enrichment of extender with carnitine improved buffalo sperm quality by increasing ATP generation and modulating ROS production, without affecting in vitro fertilizing ability.
Animals
Antioxidant supplementation has been proposed as a new strategy to improve the long-term preserva... more Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 2...
Theriogenology, 2013
The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycopr... more The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosomereacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro-matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN þ heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo.
Animals : an Open Access Journal from MDPI, 2021
Simple Summary Suboptimal fertility in males accounts for about two-thirds of infertility cases, ... more Simple Summary Suboptimal fertility in males accounts for about two-thirds of infertility cases, thus being of serious concern for the dairy industry, where optimal fertility is fundamental for farm profitability. Although genetic defects responsible for subfertility have been identified, the role of seminal compounds on fertility remain unclear. Feeding rumen-protected isomers of conjugated linoleic acid (CLA) to dairy cows reportedly enhances circulating insulin-like growth factor I (IGF-I) levels. In breeding bulls, the IGF-I concentration in seminal plasma has been positively correlated with fertility rates. Therefore, the objective of the study was to evaluate the effect of dietary CLA supplementation and of their inclusion to the semen extender on bovine semen quality and freezability. Abstract Isomers of conjugated linoleic acid (CLA) enhances circulating insulin-like growth factor I (IGF-I) levels. Furthermore, fertility rate of breeding bulls is positively correlated to sem...
The improvement of fertility is an important contribution that applied scientific research can pr... more The improvement of fertility is an important contribution that applied scientific research can provide to buffalo breeding, especially in productive areas like the Campania region. Many studies have been conducted to improve the management of the female reproductive system, while insufficient are the information about the effect of the male on the reproductive efficiency in this species. Buffalo is a photoperiodic animal that tends to increase the reproductive activity during periods when daylight hours decrease. In particular, in the male, it is manifested by a reduction of libido when daylight hours increase. This causes a reduction in the number of jumps and a worsening of the qualitative characteristics of the semen produced . Also, the cryopreservation process influences the quality of the semen causing premature sperm capacitation hence, reducing its longevity in the female reproductive tract. At present, for cryopreservation of semen, standard protocols are used equally for a...
Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among the... more Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among them, translocations present a specific meiotic segregation pattern able to give a higher percentage of unbalanced gametes that can induce fertility problems. In this study, the meiotic segregation patterns of 1p, 1q and 18 Bubalus bubalis chromosomes were analyzed in both total sperm fraction and motile sperm fraction of a t(1p;18) carrier and a control bulls by triple-color FISH analysis with a pool of specific BAC probes. The frequencies of each total sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 39%, 20%, 1% and 38%, respectively. On the other hand, the frequencies of each motile sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 93%, 5%, 0% and 2%, respectively. The frequencies of normal sperms in the carrier were 27% and 69% in total sperm fraction and mot...
Italian Journal of Animal Science
Reproduction in Domestic Animals
The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibito... more The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 h with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (P<0.05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4 respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (P<0.05), while the percentage of spermatozoa with high ΔΨm increased (P<0.05). The results of Experiment 2 showed that 1 h-treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (P<0.05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (P<0.05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.
Animal Reproduction Science
Animals
Semen cryopreservation determines several sperm damages, including the loss of fertility-associat... more Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine...
Scientific Reports
Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the... more Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the oocyte molecular status in relation to season are poorly understood. This study characterizes the microRNA (miRNA) and transcriptomic profiles of oocytes (OOs) and corresponding follicular cells (FCs) from buffalo ovaries collected in the breeding (BS) and non-breeding (NBS) seasons. In the BS, cleavage and blastocyst rates are significantly higher compared to NBS. Thirteen miRNAs and two mRNAs showed differential expression (DE) in FCs between BS and NBS. DE-miRNAs target gene analysis uncovered pathways associated with transforming growth factor β (TGFβ) and circadian clock photoperiod. Oocytes cluster in function of season for their miRNA content, showing 13 DE-miRNAs between BS and NBS. Between the two seasons, 22 differentially expressed genes were also observed. Gene Ontology (GO) analysis of miRNA target genes and differentially expressed genes (DEGs) in OOs highlights pathways related to triglyceride and sterol biosynthesis and storage. Co-expression analysis of miRNAs and mRNAs revealed a positive correlation between miR-296-3p and genes related to metabolism and hormone regulation. In conclusion, season significantly affects female fertility in buffalo and impacts on oocyte transcriptomic of genes related to folliculogenesis and acquisition of oocyte competence. Water buffalo (Bubalus bubalis) is an important livestock resource for both developing and developed countries. The major factor affecting buffalo farming profitability is reproductive seasonality, resulting in cycles of calving and milk production. Buffalo is a short-day breeder, with increased fertility in response to decreasing day length 1,2. This photoperiod-dependent seasonality pattern is more pronounced as distance from the equator, together with variations in the light/dark ratio, increases. In Italy, in order to satisfy market demand, out of breeding mating strategy (OBMS), consisting in interrupting sexual promiscuity or the use of artificial insemination (AI) during the breeding season (BS), is commonly utilized 2. The OBMS improves the distribution of calving throughout the year, but it reduces fertility 3. Longer post-partum anoestrus periods as well as higher incidence of embryonic mortality are observed in months with increasing daylight length and particularly in midwinter , which coincides with the transition to seasonal anoestrus at Italian latitudes 1,4. The embryonic mortality is due to inadequate luteal growth and function, resulting in reduced progesterone secretion 5. This has a negative impact on embryo growth, associated with alterations in transcriptomic and proteomic profiles of the embryos and chorioamnios/caruncles 6,7 , which ultimately impair embryo attachment to the uterine endometrium.
Animals
The effect of crocin in the semen extender before cryopreservation was evaluated on sperm paramet... more The effect of crocin in the semen extender before cryopreservation was evaluated on sperm parameters of 20 bucks of five different breeds: Garganica (GA), Jonica (JO), Maltese (MA), Mediterranean Red (MR) and Saanen (SA). Semen samples were centrifuged, to remove seminal plasma, divided in two aliquots and diluted with Tris-egg-yolk-based extender, containing 0 (control group) and 1 mM crocin. Crocin concentration was established after a preliminary dose trial. On fresh and frozen-thawed sperm, motility, viability, morphology, membrane integrity, DNA fragmentation and ROS levels were evaluated. The freezing process led to a decrease (p < 0.05) in all the sperm parameters recorded, confirming the deleterious effect of cryopreservation on goat semen. The most interesting result regarding the inclusion of crocin in the extender before cryopreservation was as follows: Crocin significantly improved (p < 0.05) sperm motility in all breeds, except for Mediterranean Red, compared to t...
Italian Journal of Animal Science
The aim of this study was to determine whether buffalo heifers maintained at a constant live weig... more The aim of this study was to determine whether buffalo heifers maintained at a constant live weight (LW) and body condition score (BCS) by a restricted diet show changes in reproductive function in response to changes in day length. Heifers were fed a low energy diet (86% daily intake recommended for the species) throughout the study, which involved an 8-week period of decreasing day length and a 7-week period of increasing day length. Weekly, LW, BCS and ovarian follicular/oocyte population were assessed. Blood and follicular fluid samples were taken at the time of ovum pickup for hormone analyses and at 30-day intervals to measure metabolic substrates. Buffalo heifers maintained LW and BCS throughout the study and blood concentrations of the main metabolic hormones, such as GH, insulin, IGF-1 and thyroid hormones were not influenced by photoperiod. Likewise, there were no differences in metabolic substrates such as glucose, NEFA, triglycerides and HDL cholesterol, suggesting that the metabolic condition remained essentially the same. During increasing day length periods, the number of total follicles (8.4 ± 0.5 vs. 11.9 ± 0.6; p < .01), cumulus-oocyte complexes (2.2 ± 0.3 vs 4.3 ± 0.5; p < .01) and superior quality Grade A þ B COCs (0.2 ± 0.1 vs 1.1 ± 0.2; p < .01) decreased. These features were associated to reduced oestradiol levels both in plasma (3.9 ± 0.4 vs 9.2 ± 0.5 pg/mL; p < .01) and follicular fluid (49.7 ± 12.9 vs. 109.1 ± 25.8 ng/mL; p ¼ .05), as well as to reduced intrafollicular IGF-1 levels (45.5 ± 4.0 vs. 58.5 ± 3.9 ng/mL; p < .05). These findings suggest that the ovarian function in buffalo heifers is influenced by photoperiod independent of nutritional and metabolic status. HIGHLIGHTS Metabolic profile, LW and BCS do not change in buffalo heifers on a restricted diet Photoperiod affects ovarian function in heifers regardless of metabolic status Follicular and oocyte population decrease in increasing day length periods Oestradiol and IGF-1 levels in follicular fluid are affected by photoperiod ARTICLE HISTORY
Italian Journal of Animal Science
The aim of this work was to evaluate whether the residual amount of c-glutamyl-transferase (GGT) ... more The aim of this work was to evaluate whether the residual amount of c-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (r s ¼ .4; p < .05) both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage (r s ¼ .5; p < .05 and .8; p < .01, for sperm and supernatant respectively) and blastocyst (r s ¼ .6 and .9, for sperm and supernatant respectively; p < .01) rates obtained after IVF. These results suggest that GGT could play an important role in the protection of sperm against oxidative stress and could be considered a reliable marker to assess frozen/thawed sperm quality in bovine.
Theriogenology, 2016
The aim of this study was to evaluate the effect of resveratrol supplementation of semen extender... more The aim of this study was to evaluate the effect of resveratrol supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. After the initial semen assessment, buffalo semen was cryopreserved in BioXcell containing 0 (control group), 0.5, 1, 10, and 50-μM resveratrol. After thawing, viability, motility, and capacitation status (assessed by localization of phosphotyrosine-containing proteins) were evaluated. Based on the results of the dose-response trial, the concentration of 50 μM was selected for further assessments, such as membrane integrity, total antioxidant capacity, reactive oxygen species, and lipid peroxidation (LPO) levels. Moreover, in vitro fertilizing ability by heterologous IVF and in vivo fertility were assessed. No differences among groups were recorded in sperm motility and viability (on average 52.3 ± 2.1% and 76.6 ± 1.3%, respectively). However, data showed a resveratrol dose-dependent effect on sperm capacitation status, with a significant reduction of the cryopreservation-induced capacitation with the higher concentrations tested. In particular, both 10- and 50-μM resveratrol increased (P < 0.01) the percentage of sperm displaying pattern A (low capacitation level), but treatment with 50-μM resveratrol also decreased (P < 0.01) the proportion of sperm exhibiting pattern EA (high-capacitation level) compared with the control. Interestingly, supplementation of semen extender with resveratrol increased membrane integrity, indicated by the higher percentage of hypo-osmotic swelling positive sperm (55.6 ± 0.6 vs. 48.4 ± 0.7; P < 0.01), and total antioxidant capacity (1.36 ± 0.01 vs. 1.32 ± 0.02 mM/L; P < 0.05) compared with the control. Intracellular reactive oxygen species decreased in resveratrol-treated sperm compared with the control, as indicated by dihydroethidium values (0.17 ± 0.01 and 0.22 ± 0.01 μM/μL dihydroethidium, respectively; P < 0.01). Moreover, when IVF was carried out by using semen treated with 50 μM resveratrol, the normal fertilization rate considerably improved (60.8%, P < 0.05) compared with the control (51.3%). However, no differences were recorded in pregnancy rates at 60 days post-AI with resveratrol-treated semen (50 μM) compared with the control (48.7 vs. 46.5%, respectively). In conclusion, the inclusion of 50-μM resveratrol in the extender decreases capacitation-like changes and oxidative stress, improving membrane stability and in vitro fertilizing ability of buffalo semen.
Italian Journal of Animal Science, Nov 21, 2019
The study evaluated the effects of eCG treatment prior to ovum pick up (OPU) on follicular popula... more The study evaluated the effects of eCG treatment prior to ovum pick up (OPU) on follicular population, oocyte and embryo yields in summer and autumn in Podolic cattle. The effects of repeated OPU on cattle wellbeing was also documented. Twenty-six animals were used, and split into two groups, treatment (OPU; n ¼ 18) and control (CG; n ¼ 8). The OPU cattle were subsequently split into two subgroups (n ¼ 9) and underwent repeated OPU, without and with eCG priming, for a total of 8 sessions (4 sessions/season). Follicular population, oocyte and embryo yields were recorded in those subgroups. CG was handled in the same manner of OPU, except for epidural anaesthesia and follicular aspiration. Biochemical profile, serum protein electrophoresis and haptoglobin levels were analysed in OPU and CG. Hormonal priming increased the number of medium follicles (1.7 ± 0.2 vs 0.9 ± 0.2, p < .05), while it decreased the recovery rate and number of cumulus-enclosed oocytes (COCs) (recovery rate: 38.3 ± 3.5 vs 60.5 ± 4.0%; COCs: 2.3 ± 0.3 vs 3.4 ± 0.4, respectively; p < .01). However, priming increased cleavage (72.9 ± 5.7 vs 49.4 ± 5.4; p < .05) and blastocyst (41.1 ± 5.7 vs 23.0 ± 4.2; p ¼ .054) rates. With regard to season's effect, a higher number of COCs was recorded in autumn than in summer (3.1 ± 0.4 vs 2.6 ± 0.3; p < .05) without affecting though the number of embryos produced (0.9 on average). Since haematological parameters did not vary between OPU and CG, our preliminary data suggest that repeated OPU may be used as a conservation strategy in Podolic cattle without affecting wellbeing. HIGHLIGHTS Podolic cattle is an endangered Italian breed, reared in semi-extensive/extensive systems Ovum pickup (OPU) can be carried out in this breed without impairing animal health and welfare The eCG treatment before OPU (priming) did not improve the number of embryos produced per donor.
Animal Reproduction Science
Scientific Reports, Sep 1, 2022
In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one ... more In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one of the major factors affecting farming profitability. Follicular fluid (FF) contains extracellular vesicles (EVs) playing an important role in modulating oocyte developmental competence and carrying microRNAs (miRNAs) essential for in vitro fertilization outcomes. The aim of this work was to characterize the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) follicles collected in the breeding (BS) and non-breeding (NBS) seasons, to unravel the molecular causes of the reduced oocyte competence recorded in buffalo during the NBS. In total, 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) were found. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR < 0.05) between An and pO groups. A subset of the most significant DE-miRNAs between An and pO groups targets genes which function is related to the lipid and steroid metabolism, response to glucocorticoid and oestradiol stimulus. Comparison between BS and NBS showed 14 and 12 DE-miRNAs in An-FF-EVs and pO-FF-EVs, which regulate IL6 release and cellular adhesion, respectively. In conclusion, these results demonstrated that the miRNA cargo of buffalo FF-EVs varies in relation to both follicular development and season. The importance of buffalo (Bubalus bubalis) breeding is clearly indicated by the positive growth trend all over the world (Faostat.fao.org/faostat), due to specific features making this species a valuable protein source, particularly for tropical countries. A peculiar situation is described in Italy, where the success of buffalo breeding is closely related to the production of mozzarella cheese, highly requested around the world. A major limiting factor is reproductive seasonality, impeding continuity of milk production throughout the year, and hence not allowing to meet the market demand. Buffalo is a short-day breeder, with an increased reproductive activity observed during decreasing day length months 1,2. In Italy, where the Italian Mediterranean buffalo breed has been selected, the seasonality pattern shows an opposite trend to the market request. Therefore, the out of breeding mating strategy (OBMS), has been efficiently applied to distribute calving more evenly during the year 2. However, forcing buffalo cows to conceive during the non-breeding season (NBS) may lead to extended post-partum anestrus, higher rates of embryonic mortality and overall reduced fertility 1,3,4. In previous studies it was demonstrated that embryonic mortality is in part caused by impaired luteal function, and consequently reduced progesterone secretion 5. This in turn interferes with embryo growth that is accompanied by transcriptomic and proteomic changes at the level of embryos and chorioamnios/caruncles 6,7 , definitively hampering embryo attachment. Embryonic mortality during the NBS is also in part due to reduced oocyte developmental competence, as shown by the decreased cleavage and blastocyst rates obtained after in vitro fertilization during increasing daylight months in Italian Mediterranean buffaloes 8,9. A seasonal effect on follicular population and embryo outcomes was also reported by other authors 10,11. A poorer oocyte quality was observed in Murrah buffalo heifers during
Theriogenology, 2017
The aim of this study was to evaluate the effect of carnitine supplementation of semen extender o... more The aim of this study was to evaluate the effect of carnitine supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. Buffalo semen was cryopreserved in BioXcell containing 0 (control group), 2.5 and 7.5-mM carnitine. After thawing, viability, motility, membrane integrity and capacitation status (assessed by localization of phosphotyrosine-containing proteins and chlortetracycline, chlortetracycline assay) were evaluated. Furthermore, total antioxidant capacity, reactive oxygen species (ROS) and lipid peroxidation levels, as well as adenosine triphosphate (ATP) content and phospholipids concentration were assessed. Finally, in vitro-fertilizing ability was evaluated after heterologous IVF. An increased post-thawing sperm motility and membrane integrity were recorded in both treated groups compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6%; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.05 and 48.44 ± 0.69, 55.19 ± 0.54, 59.63 ± 0.30%; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01 with 0, 2.5-mM, and 7.5-mM carnitine, respectively). Supplementation of carnitine to the freezing extender decreased (P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01) the percentage of sperm displaying fluorescence at both equatorial and anterior acrosomal regions (pattern EA), corresponding to high capacitation level, compared with the control (30.3 ± 3.8, 18.8 ± 2.8, and 7.2 ± 2.9%, respectively, with 0, 2.5-mM, and 7.5-mM carnitine). In agreement with this, carnitine also decreased (P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01) the percentage of sperm displaying chlortetracycline pattern B (capacitated sperm) (63.8 ± 1.8, 46.8 ± 2.2, and 37.2 ± 1.8%, respectively with 0, 2.5-, and 7.5-mM carnitine). Interestingly, carnitine increased total antioxidant capacity and ATP content of buffalo frozen-thawed sperm (1.32 ± 0.02, 1.34 ± 0.01, 1.37 ± 0.01 mM/L and 4.1 ± 0.1, 5.3 ± 0.1 and 8.2 ± 0.4 nM × 10(8) sperm; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01, respectively, with 0, 2.5- and 7.5-mM carnitine). Intracellular ROS decreased in carnitine-treated sperm compared with the control, as indicated by dihydroethidium (DHE) values (0.22 ± 0.01, 0.18 ± 0.01, and 0.14 ± 0.0 μM/100 μL dihydroethidium, respectively, with 0, 2.5-, and 7.5-mM carnitine; P &amp;amp;amp;amp;amp;amp;amp;lt; 0.01), whereas lipid peroxidation levels (on average 30.5 ± 0.3 nmol/mL MDA) and phospholipids concentration (on average 0.14 ± 0.00 μg/120 × 10(6) sperm) were unaffected. Despite the improved sperm quality, the percentage of normospermic penetration after IVF was not influenced (on average 53.5 ± 1.8). In conclusion, enrichment of extender with carnitine improved buffalo sperm quality by increasing ATP generation and modulating ROS production, without affecting in vitro fertilizing ability.
Animals
Antioxidant supplementation has been proposed as a new strategy to improve the long-term preserva... more Antioxidant supplementation has been proposed as a new strategy to improve the long-term preservation of semen. The aim of this study was to evaluate the effect of Maca supplementation of semen extender on quality-related canine semen parameters during cooling. Ejaculates from nine dogs were cooled for 7 days in the absence (control group) or in the presence of 10, 20 and 50 μL/mL of an aqueous extract of Maca. Sperm were evaluated for sperm viability, motility, DNA fragmentation and lipid peroxidation after 3 h, 24 h, 4 days and 7 days of storage. The addition of 10 μL/mL of Maca preserved sperm DNA and plasma membrane integrity at 3 h and increased sperm curvilinear velocity after 24 h. Treatment with 20 and 50 μL/mL of Maca increased the percentage of hyperactivated sperm after 3 h. Moreover, semen treated with 20 μL/mL of Maca decreased lipid peroxidation at 24 h. A significant reduction of sperm DNA and plasma membrane integrity as well as of kinetics parameters between 3 and 2...
Theriogenology, 2013
The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycopr... more The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosomereacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro-matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN þ heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo.
Animals : an Open Access Journal from MDPI, 2021
Simple Summary Suboptimal fertility in males accounts for about two-thirds of infertility cases, ... more Simple Summary Suboptimal fertility in males accounts for about two-thirds of infertility cases, thus being of serious concern for the dairy industry, where optimal fertility is fundamental for farm profitability. Although genetic defects responsible for subfertility have been identified, the role of seminal compounds on fertility remain unclear. Feeding rumen-protected isomers of conjugated linoleic acid (CLA) to dairy cows reportedly enhances circulating insulin-like growth factor I (IGF-I) levels. In breeding bulls, the IGF-I concentration in seminal plasma has been positively correlated with fertility rates. Therefore, the objective of the study was to evaluate the effect of dietary CLA supplementation and of their inclusion to the semen extender on bovine semen quality and freezability. Abstract Isomers of conjugated linoleic acid (CLA) enhances circulating insulin-like growth factor I (IGF-I) levels. Furthermore, fertility rate of breeding bulls is positively correlated to sem...
The improvement of fertility is an important contribution that applied scientific research can pr... more The improvement of fertility is an important contribution that applied scientific research can provide to buffalo breeding, especially in productive areas like the Campania region. Many studies have been conducted to improve the management of the female reproductive system, while insufficient are the information about the effect of the male on the reproductive efficiency in this species. Buffalo is a photoperiodic animal that tends to increase the reproductive activity during periods when daylight hours decrease. In particular, in the male, it is manifested by a reduction of libido when daylight hours increase. This causes a reduction in the number of jumps and a worsening of the qualitative characteristics of the semen produced . Also, the cryopreservation process influences the quality of the semen causing premature sperm capacitation hence, reducing its longevity in the female reproductive tract. At present, for cryopreservation of semen, standard protocols are used equally for a...
Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among the... more Chromosomal aberrations are relatively frequent pathologies in both humans and animals. Among them, translocations present a specific meiotic segregation pattern able to give a higher percentage of unbalanced gametes that can induce fertility problems. In this study, the meiotic segregation patterns of 1p, 1q and 18 Bubalus bubalis chromosomes were analyzed in both total sperm fraction and motile sperm fraction of a t(1p;18) carrier and a control bulls by triple-color FISH analysis with a pool of specific BAC probes. The frequencies of each total sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 39%, 20%, 1% and 38%, respectively. On the other hand, the frequencies of each motile sperm fraction products in the carrier resulting from alternate, adjacent I, adjacent II and 3:1 segregation were 93%, 5%, 0% and 2%, respectively. The frequencies of normal sperms in the carrier were 27% and 69% in total sperm fraction and mot...
Italian Journal of Animal Science
Reproduction in Domestic Animals
The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibito... more The aim of this study was to evaluate the treatment of bovine semen with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), before or after freezing on semen quality. After the initial assessment, sperm from 4 bulls were pooled (Experiment 1) and cryopreserved in BioXcell containing 0, 20 and 100 μM Z-VAD-FMK. After thawing semen viability, motility, membrane integrity, as well as DNA fragmentation and ΔΨm were evaluated. In Experiment 2, bovine frozen/thawed sperm were incubated for 1 h with 0, 20 and 100 µM Z-VAD-FMK before assessing the semen quality. The treatment with Z -VAD-FMK before cryopreservation improved post-thawing sperm motility compared to the control group (P<0.05), while no differences were recorded in sperm viability and membrane integrity among groups (on average 86.8 ± 1.5 and 69.1 ± 1.4 respectively). Interestingly, at the highest concentration, DNA fragmentation decreased (P<0.05), while the percentage of spermatozoa with high ΔΨm increased (P<0.05). The results of Experiment 2 showed that 1 h-treatment with Z-VAD-FMK did not affect sperm motility and viability (on average 63.4 ± 5.8 and 83.7.1 ± 1.2, respectively). However, Z-VAD-FMK improved sperm membrane integrity (P<0.05) and at the highest concentration tested decreased the proportion of sperm showing DNA fragmentation (P<0.05). No differences were recorded in the percentage of spermatozoa with high ΔΨm (on average 57.0 ± 11.4). In conclusion, the treatment with 100 µM of the caspase inhibitor Z-VAD-FMK before freezing increased bovine sperm mass motility and ΔΨm, while decreasing sperm DNA fragmentation. Treatment of semen after thawing with 100 µM Z-VAD-FMK improved sperm membrane integrity and reduced DNA fragmentation.
Animal Reproduction Science
Animals
Semen cryopreservation determines several sperm damages, including the loss of fertility-associat... more Semen cryopreservation determines several sperm damages, including the loss of fertility-associated proteins. The purpose of the study was to compare the metabolite contents in bovine sperm and seminal plasma before and after cryopreservation, and between high- and low-fertility bulls in vitro. Forty-eight ejaculates, collected from eight bulls (six per bull), were analyzed by liquid chromatography–mass spectrometry. Cryopreservation resulted in an over-expression of lysophosphatidylcholine (0:0/18:2(9Z,12Z)) in seminal plasma. In addition, higher levels of glycine betaine and pyro-l-glutaminyl-l-glutamine were observed in cryopreserved compared to fresh spermatozoa. The fresh seminal plasma of high-fertility bulls showed an over-expression of l-acetylcarnitine, glycerol tripropanoate, 2,3-diacetoxypropyl stearate and glycerophosphocholine, and an under-expression of lysophosphatidylcholine and butyrylcarnitine, compared to low-fertility bulls. Higher levels of glycerophosphocholine...
Scientific Reports
Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the... more Season clearly influences oocyte competence in buffalo (Bubalus bubalis); however, changes in the oocyte molecular status in relation to season are poorly understood. This study characterizes the microRNA (miRNA) and transcriptomic profiles of oocytes (OOs) and corresponding follicular cells (FCs) from buffalo ovaries collected in the breeding (BS) and non-breeding (NBS) seasons. In the BS, cleavage and blastocyst rates are significantly higher compared to NBS. Thirteen miRNAs and two mRNAs showed differential expression (DE) in FCs between BS and NBS. DE-miRNAs target gene analysis uncovered pathways associated with transforming growth factor β (TGFβ) and circadian clock photoperiod. Oocytes cluster in function of season for their miRNA content, showing 13 DE-miRNAs between BS and NBS. Between the two seasons, 22 differentially expressed genes were also observed. Gene Ontology (GO) analysis of miRNA target genes and differentially expressed genes (DEGs) in OOs highlights pathways related to triglyceride and sterol biosynthesis and storage. Co-expression analysis of miRNAs and mRNAs revealed a positive correlation between miR-296-3p and genes related to metabolism and hormone regulation. In conclusion, season significantly affects female fertility in buffalo and impacts on oocyte transcriptomic of genes related to folliculogenesis and acquisition of oocyte competence. Water buffalo (Bubalus bubalis) is an important livestock resource for both developing and developed countries. The major factor affecting buffalo farming profitability is reproductive seasonality, resulting in cycles of calving and milk production. Buffalo is a short-day breeder, with increased fertility in response to decreasing day length 1,2. This photoperiod-dependent seasonality pattern is more pronounced as distance from the equator, together with variations in the light/dark ratio, increases. In Italy, in order to satisfy market demand, out of breeding mating strategy (OBMS), consisting in interrupting sexual promiscuity or the use of artificial insemination (AI) during the breeding season (BS), is commonly utilized 2. The OBMS improves the distribution of calving throughout the year, but it reduces fertility 3. Longer post-partum anoestrus periods as well as higher incidence of embryonic mortality are observed in months with increasing daylight length and particularly in midwinter , which coincides with the transition to seasonal anoestrus at Italian latitudes 1,4. The embryonic mortality is due to inadequate luteal growth and function, resulting in reduced progesterone secretion 5. This has a negative impact on embryo growth, associated with alterations in transcriptomic and proteomic profiles of the embryos and chorioamnios/caruncles 6,7 , which ultimately impair embryo attachment to the uterine endometrium.
Animals
The effect of crocin in the semen extender before cryopreservation was evaluated on sperm paramet... more The effect of crocin in the semen extender before cryopreservation was evaluated on sperm parameters of 20 bucks of five different breeds: Garganica (GA), Jonica (JO), Maltese (MA), Mediterranean Red (MR) and Saanen (SA). Semen samples were centrifuged, to remove seminal plasma, divided in two aliquots and diluted with Tris-egg-yolk-based extender, containing 0 (control group) and 1 mM crocin. Crocin concentration was established after a preliminary dose trial. On fresh and frozen-thawed sperm, motility, viability, morphology, membrane integrity, DNA fragmentation and ROS levels were evaluated. The freezing process led to a decrease (p < 0.05) in all the sperm parameters recorded, confirming the deleterious effect of cryopreservation on goat semen. The most interesting result regarding the inclusion of crocin in the extender before cryopreservation was as follows: Crocin significantly improved (p < 0.05) sperm motility in all breeds, except for Mediterranean Red, compared to t...
Italian Journal of Animal Science
The aim of this study was to determine whether buffalo heifers maintained at a constant live weig... more The aim of this study was to determine whether buffalo heifers maintained at a constant live weight (LW) and body condition score (BCS) by a restricted diet show changes in reproductive function in response to changes in day length. Heifers were fed a low energy diet (86% daily intake recommended for the species) throughout the study, which involved an 8-week period of decreasing day length and a 7-week period of increasing day length. Weekly, LW, BCS and ovarian follicular/oocyte population were assessed. Blood and follicular fluid samples were taken at the time of ovum pickup for hormone analyses and at 30-day intervals to measure metabolic substrates. Buffalo heifers maintained LW and BCS throughout the study and blood concentrations of the main metabolic hormones, such as GH, insulin, IGF-1 and thyroid hormones were not influenced by photoperiod. Likewise, there were no differences in metabolic substrates such as glucose, NEFA, triglycerides and HDL cholesterol, suggesting that the metabolic condition remained essentially the same. During increasing day length periods, the number of total follicles (8.4 ± 0.5 vs. 11.9 ± 0.6; p < .01), cumulus-oocyte complexes (2.2 ± 0.3 vs 4.3 ± 0.5; p < .01) and superior quality Grade A þ B COCs (0.2 ± 0.1 vs 1.1 ± 0.2; p < .01) decreased. These features were associated to reduced oestradiol levels both in plasma (3.9 ± 0.4 vs 9.2 ± 0.5 pg/mL; p < .01) and follicular fluid (49.7 ± 12.9 vs. 109.1 ± 25.8 ng/mL; p ¼ .05), as well as to reduced intrafollicular IGF-1 levels (45.5 ± 4.0 vs. 58.5 ± 3.9 ng/mL; p < .05). These findings suggest that the ovarian function in buffalo heifers is influenced by photoperiod independent of nutritional and metabolic status. HIGHLIGHTS Metabolic profile, LW and BCS do not change in buffalo heifers on a restricted diet Photoperiod affects ovarian function in heifers regardless of metabolic status Follicular and oocyte population decrease in increasing day length periods Oestradiol and IGF-1 levels in follicular fluid are affected by photoperiod ARTICLE HISTORY
Italian Journal of Animal Science
The aim of this work was to evaluate whether the residual amount of c-glutamyl-transferase (GGT) ... more The aim of this work was to evaluate whether the residual amount of c-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) in bovine sperm after freezing/thawing is correlated with fertility parameters, including blastocyst rates after in vitro fertilisation (IVF). The enzyme activities were determined in both spermatozoa and supernatant after centrifugation. While ALP was only correlated with sperm viability, GGT activity was correlated with sperm motility (r s ¼ .4; p < .05) both in sperm and supernatant. Interestingly, GGT activity was also correlated with cleavage (r s ¼ .5; p < .05 and .8; p < .01, for sperm and supernatant respectively) and blastocyst (r s ¼ .6 and .9, for sperm and supernatant respectively; p < .01) rates obtained after IVF. These results suggest that GGT could play an important role in the protection of sperm against oxidative stress and could be considered a reliable marker to assess frozen/thawed sperm quality in bovine.
Theriogenology, 2016
The aim of this study was to evaluate the effect of resveratrol supplementation of semen extender... more The aim of this study was to evaluate the effect of resveratrol supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. After the initial semen assessment, buffalo semen was cryopreserved in BioXcell containing 0 (control group), 0.5, 1, 10, and 50-μM resveratrol. After thawing, viability, motility, and capacitation status (assessed by localization of phosphotyrosine-containing proteins) were evaluated. Based on the results of the dose-response trial, the concentration of 50 μM was selected for further assessments, such as membrane integrity, total antioxidant capacity, reactive oxygen species, and lipid peroxidation (LPO) levels. Moreover, in vitro fertilizing ability by heterologous IVF and in vivo fertility were assessed. No differences among groups were recorded in sperm motility and viability (on average 52.3 ± 2.1% and 76.6 ± 1.3%, respectively). However, data showed a resveratrol dose-dependent effect on sperm capacitation status, with a significant reduction of the cryopreservation-induced capacitation with the higher concentrations tested. In particular, both 10- and 50-μM resveratrol increased (P < 0.01) the percentage of sperm displaying pattern A (low capacitation level), but treatment with 50-μM resveratrol also decreased (P < 0.01) the proportion of sperm exhibiting pattern EA (high-capacitation level) compared with the control. Interestingly, supplementation of semen extender with resveratrol increased membrane integrity, indicated by the higher percentage of hypo-osmotic swelling positive sperm (55.6 ± 0.6 vs. 48.4 ± 0.7; P < 0.01), and total antioxidant capacity (1.36 ± 0.01 vs. 1.32 ± 0.02 mM/L; P < 0.05) compared with the control. Intracellular reactive oxygen species decreased in resveratrol-treated sperm compared with the control, as indicated by dihydroethidium values (0.17 ± 0.01 and 0.22 ± 0.01 μM/μL dihydroethidium, respectively; P < 0.01). Moreover, when IVF was carried out by using semen treated with 50 μM resveratrol, the normal fertilization rate considerably improved (60.8%, P < 0.05) compared with the control (51.3%). However, no differences were recorded in pregnancy rates at 60 days post-AI with resveratrol-treated semen (50 μM) compared with the control (48.7 vs. 46.5%, respectively). In conclusion, the inclusion of 50-μM resveratrol in the extender decreases capacitation-like changes and oxidative stress, improving membrane stability and in vitro fertilizing ability of buffalo semen.
Italian Journal of Animal Science, Nov 21, 2019
The study evaluated the effects of eCG treatment prior to ovum pick up (OPU) on follicular popula... more The study evaluated the effects of eCG treatment prior to ovum pick up (OPU) on follicular population, oocyte and embryo yields in summer and autumn in Podolic cattle. The effects of repeated OPU on cattle wellbeing was also documented. Twenty-six animals were used, and split into two groups, treatment (OPU; n ¼ 18) and control (CG; n ¼ 8). The OPU cattle were subsequently split into two subgroups (n ¼ 9) and underwent repeated OPU, without and with eCG priming, for a total of 8 sessions (4 sessions/season). Follicular population, oocyte and embryo yields were recorded in those subgroups. CG was handled in the same manner of OPU, except for epidural anaesthesia and follicular aspiration. Biochemical profile, serum protein electrophoresis and haptoglobin levels were analysed in OPU and CG. Hormonal priming increased the number of medium follicles (1.7 ± 0.2 vs 0.9 ± 0.2, p < .05), while it decreased the recovery rate and number of cumulus-enclosed oocytes (COCs) (recovery rate: 38.3 ± 3.5 vs 60.5 ± 4.0%; COCs: 2.3 ± 0.3 vs 3.4 ± 0.4, respectively; p < .01). However, priming increased cleavage (72.9 ± 5.7 vs 49.4 ± 5.4; p < .05) and blastocyst (41.1 ± 5.7 vs 23.0 ± 4.2; p ¼ .054) rates. With regard to season's effect, a higher number of COCs was recorded in autumn than in summer (3.1 ± 0.4 vs 2.6 ± 0.3; p < .05) without affecting though the number of embryos produced (0.9 on average). Since haematological parameters did not vary between OPU and CG, our preliminary data suggest that repeated OPU may be used as a conservation strategy in Podolic cattle without affecting wellbeing. HIGHLIGHTS Podolic cattle is an endangered Italian breed, reared in semi-extensive/extensive systems Ovum pickup (OPU) can be carried out in this breed without impairing animal health and welfare The eCG treatment before OPU (priming) did not improve the number of embryos produced per donor.