Structural changes of in vitro matured buffalo and bovine oocytes following cryopreservation.

The aim of this work was to evaluate chromatin and spindle organization of buffalo and bovine in vitro matured oocytes after vitrification/warming by Cryotop and after their exposure to cryoprotectants (CP). In vitro matured oocytes were vitrified/warmed and exposed to the vitrification/warming solutions containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose as CP. Two hours after warming, oocytes were fixed and immunostained for microtubules and nuclei and examined by fluorescence microscopy. Data were analyzed by Chi Square test. A higher percentage of Telophase II stage oocytes was found in the toxicity (26 and 34% in bovine and buffalo) and the vitrification groups (13 and 7% in bovine and buffalo) compared to the control, indicating occurrence of activation. An increased percentage of oocytes with abnormal spindle and chromosome organization was found in oocytes exposed to CP (24 and 13% in bovine; 32 and 30% in buffalo respectively) and in those vitrified (26 and 31% in bovine; 26 and 29% in buffalo respectively) compared to the control (0 in bovine and 2.5 % in buffalo)

Effects of dietary supplementation of conjugated linoleic acids and their inclusion in semen extenders on bovine sperm quality

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 seminal plasma IGF-I concentration. Our objective was to evaluate the effect of dietary CLA supplementation and inclusion to the semen extender on bovine semen quality and freezability. Fourteen bulls, randomly assigned to control (CTL) and CLA (50 g/day) groups, were supplemented for 10 weeks. Samples were collected at Weeks −2 (before supplementation), 0, 4, 6 (during supplementation), 10, and 11 (after supplementation). Blood and seminal plasma were analyzed for IGF-I; the ejaculates were frozen in the following subgroups: CTL (no addition to semen extender), CLA c9, t11 (50 µM), CLA c9, t11 (100 µM), CLA t10, c12 (50 µM), CLA t10, c12 (100 µM), and CLA mix (50 µM each of CLA c9, t11 and CLA t10, c12). Sperm motility, morphology, viability, mitochondrial membrane potential, and reactive oxidative species were assessed. CLA supplementation decreased ejaculates’ total volume, increased sperm concentration, beat cross frequency, and decreased oxidative stress; it also increased plasma and seminal plasma IGF-I levels compared to the CTL. The inclusion of CLA c9, t11 100 µM and CLA mixture in the extender increased live spermatozoa percentage post-thawing compared to other groups. Our results show a beneficial effect of CLA supplementation on semen quality; however, further studies evaluating fertilization rates are necessary to corroborate the results.The National Research Foundation South Africa/ Research and Innovation Support and Advancement and the Research and Technology fund.http://www.mdpi.com/journal/animalspm2021Production Animal Studie

Effects of dietary supplementation of conjugated linoleic acids and their inclusion in semen extenders on bovine sperm quality

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 seminal plasma IGF-I concentration. Our objective was to evaluate the effect of dietary CLA supplementation and inclusion to the semen extender on bovine semen quality and freezability. Fourteen bulls, randomly assigned to control (CTL) and CLA (50 g/day) groups, were supplemented for 10 weeks. Samples were collected at Weeks −2 (before supplementation), 0, 4, 6 (during supplementation), 10, and 11 (after supplementation). Blood and seminal plasma were analyzed for IGF-I; the ejaculates were frozen in the following subgroups: CTL (no addition to semen extender), CLA c9, t11 (50 µM), CLA c9, t11 (100 µM), CLA t10, c12 (50 µM), CLA t10, c12 (100 µM), and CLA mix (50 µM each of CLA c9, t11 and CLA t10, c12). Sperm motility, morphology, viability, mitochondrial membrane potential, and reactive oxidative species were assessed. CLA supplementation decreased ejaculates’ total volume, increased sperm concentration, beat cross frequency, and decreased oxidative stress; it also increased plasma and seminal plasma IGF-I levels compared to the CTL. The inclusion of CLA c9, t11 100 µM and CLA mixture in the extender increased live spermatozoa percentage post-thawing compared to other groups. Our results show a beneficial effect of CLA supplementation on semen quality; however, further studies evaluating fertilization rates are necessary to corroborate the results.The National Research Foundation South Africa/ Research and Innovation Support and Advancement and the Research and Technology fund.http://www.mdpi.com/journal/animalspm2021Production Animal Studie

Rumen-protected methionine supplementation alters lipid profile of preimplantation embryo and endometrial tissue of Holstein cows

Our objective is to evaluate the effects of feeding rumen-protected Met (RPM) throughout the transition period and early lactation on the lipid profile of the preimplantation embryos and the endometrial tissue of Holstein cows. Treatments consisted of feeding a total mixed ration with top-dressed RPM (Smartamine® M, Adisseo, Alpharetta, GA, United States; MET; n = 11; RPM at a rate of 0.08% of DM: Lys:Met = 2.8:1) or not (CON; n = 9, Lys:Met = 3.5:1). Endometrial biopsies were performed at 15, 30, and 73 days in milk (DIM). Prior to the endometrial biopsy at 73 DIM, preimplantation embryos were harvested via flushing. Endometrial lipid profiles were analyzed using multiple reaction monitoring-profiling and lipid profiles of embryos were acquired using matrix assisted laser desorption/ionization mass spectrometry. Relative intensities levels were used for principal component analysis. Embryos from cows in MET had greater concentration of polyunsaturated lipids than embryos from cows in CON. The endometrial tissue samples from cows in MET had lesser concentrations of unsaturated and monounsaturated lipids at 15 DIM, and greater concentration of saturated, unsaturated (specifically diacylglycerol), and monounsaturated (primarily ceramides) lipids at 30 DIM than the endometrial tissue samples from cows in CON. In conclusion, feeding RPM during the transition period and early lactation altered specific lipid classes and lipid unsaturation level of preimplantation embryos and endometrial tissue

Analisi di alcuni fattori che influenzano lo sviluppo, la distribuzione dei sessi e la resistenza alla crioconservazione di embrioni bovini e bufalini.

Negli ultimi anni, un numero sempre maggiore di aziende zootecniche ha iniziato ad avvalersi delle biotecnologie riproduttive, perché così facendo possono incrementare le performance produttive e indirizzare la produzione verso una qualità spesso impensabile, ottenendo nel contempo la riduzione dei rischi di trasmissione di malattie, tutto nell’ottica di una risposta più adeguata alle richieste di un mercato sempre più specifico. Pertanto prove sperimentali differenti sono state condotte al fine di migliorare, l’efficienza della fecondazione in vitro e la resa nella coltura embrionale in vitro nelle specie bovina ed in quella bufalina L’insieme dei risultati ha dimostrato l’esistenza di evidenti differenze nei fabbisogni metabolici degli embrioni bufalini rispetto a quelli bovini, di cui bisogna tener conto per ottimizzare il sistema in questa specie

ASSESSMENT OF VITRIFICATION-INDUCED STRUCTURAL DAMAGES IN MATURED BOVINE OOCYTES BY RAMAN MICROSPECTROSCOPY

Vitrification induces ultrastructural and structural damages in different oocyte structures such as membrane, zona pellucida (ZP) and cytoplasm.1 Recently Raman Microspectroscopy (RMS) has been used to assess the changes caused by vitrification/ warming of in vitro matured ovine oocytes.2 RMS is a noninvasive technique for studying the molecular composition of cells, based on the inelastic scattering of laser photons by vibrating molecules. Aim of the present study was to investigate the structural modifications of ZP and cytoplasm of vitrified/warmed in vitro matured bovine oocytes by RMS. Abattoir derived in vitro matured oocytes (n=171) were denuded and divided in three experimental groups: control untreated oocytes (CTR), oocytes only exposed to vitrification/warming solutions (CPA) and vitrified/warmed oocytes (VITRI). Oocytes were exposed/vitrified in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3 M). RMS analysis was carried out on CTR matured oocytes (after 22h) and on CPA and VITRI oocytes at different incubation times (0,1,2,3 and 4h) after exposure/warming. The analysis of the large spectral date set, acquired by RMS were performed by Principal Component Analysis (PCA). Our experimental outcomes suggest that vitrification induce a transformation of the protein secondary structure from the α-helices to the b-sheet form in the ZP, while lipids tend to assume a more ordered configuration. Both effects induce a mechanical stiffening of ZP, which could explain the reduced fertility of vitrified oocytes with respect to the untreated ones. Intriguingly, these transformations present a certain degree of reversibility, which renders vitrified oocytes more similar to untreated cells after 2h of warming at room temperature

Effect of bull on in vitro sperm capacitation induced by different agents in buffalo species (Bubalus bubalis)

The aim of this work was to evaluate the effect of bull on the efficacy of different capacitating agents in buffalo. Spermatozoa derived from 4 different bulls were incubated in absence of a capacitating agent, in presence of 0.01 mM heparin and in 20% buffalo estrous serum (BES) for 2 hours. Sperm were then exposed to lysophosphatidylcholine, an agent that induces acrosome reaction only on capacitated spermatozoa. A double staining technique with Trypan-blue/Giemsa was used to evaluate viability and acrosome status of spermatozoa fixed in 37% formaldehyde. The efficiency of capacitation was evaluated by assessing the percentage of acrosome-reacted (AR) spermatozoa in the treated groups. A bull effect on sperm capacitation in vitro was demonstrated, as indicated by differences in the percentage of AR sperm among bulls regardless of the treatment. In particular when heparin was used as capacitating agent bull C gave higher percentages of AR sperm than bull A (22.7 vs. 14.0%, respectively) with intermediate values for bulls B and D (16.7 and 19.1%, respectively), whereas when BES was used bull D was the most efficient one (22.1, 21.9, 25.0, and 35.0% for bulls A, B, C and D, respectively)

Effect of energy source on in vitro embryo development and freezability in cattle

Most media employed for producing bovine embryos in vitro include glucose as an energy source despite putative toxic effects. The aim of this work was to evaluate whether replacing glucose with myo-inositol and citrate during IVC improves in vitro embryo development and resistance to cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro using standard procedures. After 20–22 h of gametes co-incubation, zygotes were denuded and cultured in SOF containing either 1.5 mM glucose or 2.77 mM myo-inositol and 0.34 mM citrate for 7 days. Embryos were first incubated in 7.5 % EG and 7.5 % dimethyl sulfoxide (DMSO) for 3 min, then transferred into 16.5 % EG and 16.5 % DMSO and 0.5 M sucrose for 25 sec before being loaded into the cryotop. Warming was carried out by immerging the cryotop into a 0.25 M sucrose solution and by transferring the embryos into a 0.15 M sucrose for 5 min. Vitrified-warmed embryos were then cultured in vitro for further 24 hr, after which the embryo survival rate was recorded. Data were analyzed by Chi-Square test. The results of this study showed that myo-inositol-citrate increased blastocyst yield (37.4 vs 29.5 %, respectively; P<0.01). However, blastocysts produced in the medium containing myo-inositol and citrate had a lower survival rate after vitrification-warming than those cultured with glucose (60.4 % and 73.6 %, respectively; P<0.05). In conclusion, replacement of glucose with myo-inositol and citrate during culture increases blastocyst production without improving embryo quality, i.e. resistance to cryopreservatio

Genetic variability of Prolactin (PRL) and Prolactin Receptor (PRLR) genes in the Italian Mediterranean river buffalo.

In mammals, the hormone prolactin (PRL) is best known for its role in the regulation of lactation (Lee et al. 2007). PRL affects multiple reproductive and metabolic functions through its receptors (PRLR), characterized by the ability to activate Janus kinase 2 and signal transducer and activator of transcription (Fleenor et al. 2006). PRL is an anterior pituitary peptide hormone involved in many endocrine activities essential for reproductive success (Vaclavicek et al. 2006). Several polymorphic sites have been detected within PRL and PRLR genes, and significant associations among their variants with milk production traits have been described in dairy cattle (Alfonso et al. 2012; Zhang et al. 2008). PRL (EMBL EF054878) and PRLR (EMBL GQ339914) genes consist of 5 and 9 exons, coding for a protein of 219 and 581 amino acids, respectively. A partial sequencing of PRL exon 5 and PRLR exon 9 performed on 10 Italian Mediterranean river buffaloes allowed us to identify 1 SNP for PRL (C108→T, numbering from the 1st nt of exon 5) and 7 SNPs for PRLR (G128→A, C374→T, G517→A, C520→A, C622→T, G626→A and T707→C, numbering from the 1st nt of exon 9). The PRL SNP is silent (Leu202) as well as 626 (Ala494) and 707 (Asp521) PRLR SNPs. The other 5 PRLR SNPs are responsible of the following amino acid changes: His328→Arg, Ala410→Val, Asp458→Asn, Gln459→Lys and Thr493→Met. 574 buffaloes of several farms in Salerno province (South Italy) were genotyped in out-sourcing (http://kbioscience.co.uk) for 108 PRL and 128 PRLR SNPs. The genotype distribution of investigated population was 10 C/C, 122 C/T and 442 T/T (frequency of T allele 0.87) for PRL and 9 G/G, 108 G/A and 457 A/A for PRLR (frequency of A allele 0.89). For both SNPs, the investigated population was in Hardy–Weinberg equilibrium. Such polymorphisms could represent useful genetic markers for association studies with quali-quantitative milk characteristics, but further studies are needed to evaluate their potential use