Vitrification of pig oocytes induces changes in histone H4 acetylation and histone H3 lysine 9 methylation (H3K9)

In the present study, acetylation status of histone H4 and methylation status of the lysine 9 residue of histone H3 (H3K9) were assessed by immunofluorescence in order to determine the effect of vitrification on epigenetic status of pig MII oocytes. Hyperacetylation of H4 and dimethylation of H3K9 were assessed in control oocytes, after cryoprotectant treatment and after vitrification at two time points, immediately after warming and after a post-warming incubation for 2?h. While no changes in the immunopositivity for both the epitopes were recorded after cryoprotectants, the percentage of negative oocytes for dimethyl H3K9 was observed to increase immediately after devitrification. The influence of vitrification was more evident after 2?h post-thaw incubation when acetylation status of H4 significantly increased and a rise in the percentages of both oocytes exhibiting strong positivity and negative oocytes for dimethyl H3K9 was observed. In conclusion, acetylation of H4 and methylation of H3K9 are altered by vitrification procedure that may lead to an aberrant epigenetic presentation of female chromatin to the fertilizing event and may be, at least in part, responsible for the reduction of developmental competence of vitrified pig oocytes.     

Beta‐mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low‐molecular thiol compounds can be added to culture media. Beta‐mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n = 293, BME 0.1 n = 270, BME 0.7 n = 285) were stained with Hoechst 33342 and examined for nuclear stage after 26 hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n = 83, BME 0.1 n = 65, BME 0.7 n = 80). Cleavage rates were determined after 60 hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1 mM and 55.1% BME 0.7 mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7 mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Enhancing insemination performance in pigs through controlled release of encapsulated spermatozoa

Encapsulation of boar semen is a novel technique that allows insemination to be performed as a single intervention without the need to dilute the semen. The research reviewed in this paper shows that spermatozoa encapsulated in alginate are able to achieve the same fertility as two or three inseminations per oestrus using standard techniques and unencapsulated cells. The use of encapsulated spermatozoa is currently limited by the need for longer semen processing time and wastage of disposable material (catheters, plastic bottles, etc.). In this review, the advantages, the drawbacks and the future possibilities for artificial insemination with encapsulated spermatozoa in the sow are discussed, with the aim of applying this promising new methodology for the optimization of sow reproductive performance.     

Acyl Ghrelin and Metabolic Hormones in Pregnant and Lactating Sows

Ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor, is considered a pleiotropic regulator involved in a large array of functions, including control of energy balance, regulation of food intake and, more recently, modulation of the reproductive axis. The present study was aimed at determining the changes in plasma concentrations of acyl-ghrelin in pregnant and lactating sows, with special emphasis on the relationship with the levels of GH, leptin, non-esterified fatty acids (NEFA) and insulin-like growth factor (IGF-1). Blood samples were collected via jugular venipuncture from 22 multiparous sow 30, 60 and 90 days after artificial insemination, 7 and 21 days after farrowing and at first oestrus post-weaning. Plasma concentrations of acyl-ghrelin, leptin, GH and IGF-1 were quantified by validated radioimmunoassay; NEFA were determined using a colorimetric procedure. Plasma acyl ghrelin levels were highest at 30 days of pregnancy and decreased thereafter and during lactation. At the beginning of lactation, GH, IGF-1 and NEFA concentrations significantly increased, while a significant reduction occurred in leptin. In conclusion, ghrelin concentrations in sow maternal circulation does not seem to play an important role in maintaining circulating GH levels during lactation; moreover, ghrelin is not associated with leptin, NEFA and IGF-1 levels.     

Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure

This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir.     

Embryo Production in Superovulated Goats Treated with Insulin Before or After Mating or By Continuous Propylene Glycol Supplementation

Seventeen adult and cyclic Moxoto goats were synchronized using 60 mg MPA vaginal sponge for 11 days and 50 μg cloprostenol, 48 h before sponge removal, and superovulated with 120 mg pFSH i.m. in decreasing doses at 12 h intervals for three consecutive days. In seven goats, 0.2 IU/kg BW/day of long acting insulin was subcutaneously injected at same time as pFSH, and in the other five goats, the same dose of insulin was injected for three consecutive days starting 24 h after mating. Finally, five goats were supplemented with an oral dose of 80 ml/goat/day of propylene glycol continuously during the experiment. The animals were flushed at 7 days after mating and the embryos were classified based on International Embryo Transfer Society criteria. Blood samples were collected every 3 days for insulin assay. Administration of insulin raised the insulin levels of the goats (p < 0.05), whereas in the group treated with propylene glycol, insulin rate was different only between FSH treatment and after mating (p < 0.05). Similar rates of recovery for total (80.05 ± 9.78%) or transferable structures (61.03 ± 15.13%) were obtained. Treatment was not influenced (p > 0.05) by responsiveness to superovulation, which averaged 64%. By contrast, insulin treatments were shown to increase the number of embryos considered excellent with respect to goats supplemented with propylene glycol (p < 0.05). When insulin was given before mating, a strong relationship (r = 0. 90) (p < 0.05) between number of transferable embryo and ovulations was observed in the animals. In conclusion, superovulated goats treated with low doses of exogenous insulin resulted in an enhancement in embryo quality, which was related to changes in circulating insulin concentrations.     

Assessment of Some Morphofunctional Characteristics of Flow-Cytometrically Sorted and Stained Boar Spermatozoa

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