Reproduction Fertility and Development (REPROD FERT DEVELOP )

Publisher: Commonwealth Scientific and Industrial Research Organization (Australia); Fertility Society of Australia; Australian Academy of Science; Australian Society for Reproductive Biology; Society for Reproductive Biology, CSIRO Publishing

Description

Reproduction, Fertility and Development is an international journal for the publication of original and significant contributions related to reproduction and developmental biology in humans, domestic animals and wildlife. Contributions may take the form of research articles, reviews, short communications or viewpoint articles that deal with the scientific aspects of reproductive and developmental physiology, biochemistry, endocrinology, immunology, cell biology, genetics and behaviour, and the applications of reproductive technologies in humans, livestock, wildlife and pest management.

  • Impact factor
    2.58
    Show impact factor history
     
    Impact factor
  • 5-year impact
    2.56
  • Cited half-life
    6.80
  • Immediacy index
    0.49
  • Eigenfactor
    0.01
  • Article influence
    0.66
  • Website
    Reproduction, Fertility and Development website
  • Other titles
    Reproduction fertility and development
  • ISSN
    1031-3613
  • OCLC
    19505713
  • Material type
    Conference publication, Periodical, Internet resource
  • Document type
    Journal / Magazine / Newspaper, Internet Resource

Publisher details

CSIRO Publishing

  • Pre-print
    • Author can archive a pre-print version
  • Post-print
    • Author can archive a post-print version
  • Conditions
    • On author's personal repository or institutional repository
    • Must link to publisher version
    • Published source must be acknowledged
    • Publisher's version/PDF cannot be used
  • Classification
    ​ green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: We tested whether the reversible effects of nutrition on spermatogenesis in sexually mature sheep were mediated by Sertoli cells. Rams were fed with diets designed to achieve a 10% increase (High), no change (Maintenance) or a 10% decrease (Low) in body mass after 65 days. At the end of treatment, testes were lighter in the Low than the High group (P<0.01). The Maintenance group had intermediate values that were not significantly different from those of the other two groups. Spermatogenesis (Johnsen score) was impaired in the Low group, but normal in both other groups. There was no effect of treatment on Sertoli cell numbers, although 1% of Sertoli cells appeared to retain their ability to proliferate. By contrast, Sertoli cell function was affected by dietary treatment, as evidenced by differences between the High and Low groups (P<0.05) in the expression of seven Sertoli cell-specific genes. Under-nutrition appeared to reverse cellular differentiation leading to disruption of tight-junction morphology. In conclusion, in sexually mature sheep, reversible reductions in testis mass and spermatogenesis caused by under-nutrition were associated with impairment of basic aspects of Sertoli cell function but not with changes in the number of Sertoli cells.
    Reproduction Fertility and Development 01/2015; Published online.
  • Francisco Otero-Ferrer, Marisol Izquierdo, Alireza Fazeli, William V Holt
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    ABSTRACT: The aim of the present study was to investigate the hypothesis that parental periconception nutrition in adult seahorses affects the development and growth of their offspring. We tested the hypothesis that because seahorse embryos develop inside the male's brood pouch, manipulation of the male's diet would affect offspring growth and development independently of the female's diet. Adult males and females were fed separately with either wild-caught crustaceans or commercial aquarium diet for 1 month before conception to influence the periconception environment. Approximately 10000 offspring were obtained from four different treatment groups (Male/Wild or Male/Commercial×Female/Wild or Female/Commercial). Weights, physical dimensions and fatty acid profiles of the newborns were determined. Offspring produced when the males receiving commercial diet were mated with wild-fed females were larger (P<0.05) than those produced by wild-fed males. When both males and females were fed with commercial diet, their offspring were significantly smaller than those from the other treatment groups. When commercial diet-fed females were mated with wild-fed males, the offspring showed distortion of the snout:head length ratio. These results support the view that the preconception diet received by males and females differentially affects embryonic development.
    Reproduction Fertility and Development 12/2014;
  • Wenwen Li, Karen Goossens, Mario Van Poucke, Katrien Forier, Kevin Braeckmans, Ann Van Soom, Luc J Peelman
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    ABSTRACT: Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O2) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I_BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos.
    Reproduction Fertility and Development 12/2014;
  • Robert Rekawiecki, Magdalena Karolina Kowalik, Jan Kotwica
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    ABSTRACT: The aim of the present study was to examine the effects of luteotropic and luteolytic factors on the mRNA and protein levels of progesterone receptor isoforms A (PGRA) and B (PGRB) in the bovine endometrium. Endometrial slices from Days 6-10 and 17-20 of the oestrous cycle were treated with LH (100ngmL-1), oestradiol (E2; 1×10-8M), prostaglandin (PG) E2 (1×10-6M) and PGF2? (1×10-6M) and the nitric oxide donor NONOate (1×10-4M); these treatments lasted for 6h for mRNA expression analysis and 24h for protein expression analysis. On Days 6-10 of the oestrous cycle PGRAB (PGRAB; the entire PGRA mRNA sequence is common to the PGRB mRNA sequence) mRNA expression in endometrial slices was enhanced by E2 treatment (PPGRB mRNA expression was increased by LH (PPPPGRAB mRNA expression increased after E2 (P2 (PPGRB mRNA expression was increased by PGE2 (P2? (PPPPPP2? (P2 (P2? (P<0.001). These data suggest that luteotropic and luteolytic factors affect PGRA and PGRB mRNA and protein levels, and this may regulate the effects of progesterone on endometrial cells.
    Reproduction Fertility and Development 12/2014;
  • Thomas Kolbe, Sarjoun Sheety, Ingrid Walter, Rupert Palme, Thomas Rülicke
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    ABSTRACT: Superovulation of mice is routinely used to increase the number of obtainable ova per female. Because of the better outcome, prepubescent females are preferentially used. Here, we provide results of the impact of superovulation and mating on the wellbeing of juvenile compared with adult C57BL/6N mice. Two groups of mice (3-4 weeks vs 7-8 weeks old) were superovulated and mated. Observation of mating behaviour showed that reluctant adult females tended to fight the male's approach, whereas juveniles preferred to take flight. Faeces were collected daily for the analysis of stress hormones. There was no difference in the levels of glucocorticoid metabolites either between age groups or between treated animals and their controls. Histology after mating revealed intact vaginal mucosa without any detectable lesions in all animals regardless of age. In contrast to adults, almost all juveniles were synchronised in oestrus and produced significantly more ova. Taken together, our results reveal no increased welfare problem from using juvenile mice for superovulation and mating. Considering the higher yield of fertilisable oocytes and zygotes, it is advisable to use C57BL/6N prepubescent mice in order to reduce the number of donor females required.
    Reproduction Fertility and Development 12/2014;
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    ABSTRACT: Feral horses populate vast land areas and often induce significant ecological and economic damage throughout the landscape. Non-lethal population control methods are considered favourable in light of animal welfare, social and ethical considerations; however, no single effective, safe and species-specific contraceptive agent is currently available for use in free-ranging wild and feral horses. This review explores aspects of equine reproductive physiology that may provide avenues for the development of specific and long-lasting immunocontraceptive vaccines and some of the novel strategies that may be employed to facilitate appropriate antigen discovery in future research. Potential antigen targets pertaining to spermatozoa, the ovary and oocyte, as well as the early conceptus and its associated factors, are reviewed in the context of their suitability for immunocontraceptive vaccine development.
    Reproduction Fertility and Development 12/2014;
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    ABSTRACT: The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24h. Re-expansion rates were recorded at 3 and 24h and total cell number and apoptotic cells were determined at 24h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification-warming procedures and length of in vitro culture, as expanding and hatching-hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.
    Reproduction Fertility and Development 12/2014;
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    ABSTRACT: The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.
    Reproduction Fertility and Development 12/2014;
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    ABSTRACT: This study determined the phases of sexual development of the male Mongolian gerbil (Meriones unguiculatus) based on an integrative analysis of testicular morphology, hormonal data and sperm parameters. Male gerbils were analysed at 1, 7, 14, 21, 28, 35, 42, 50, 60, 70, 90, 100 and 120 days of age. Body, testicular and epididymal weights increased up to Day 70, 60 and 90, respectively. The impuberal phase, characterised by the presence of gonocytes, extended until Day 14. The prepubertal period lasted until Day 42, when puberty was achieved and a drastic increase in serum testosterone levels, mature adult Leydig cells and elongated spermatids was observed. Gerbils at 60 days of age showed a remarkable number of spermatozoa in the testis, epididymidis caput/corpus and cauda, and at Day 70 the maximum daily sperm production was reached. However, the gerbil may be considered sexually mature only from Day 90 onward, when sperm reserves become stable. The total transit time of spermatozoa along the epididymis of sexually mature gerbils was 11 days, with 1 day in the caput/corpus and 10 days in the cauda. These data cover a lacuna regarding the reproductive parameters of this rodent and provide foundations for its use in testicular toxicology studies.
    Reproduction Fertility and Development 12/2014;
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    ABSTRACT: The aim of this study was to evaluate the cellular changes that occur in the hamster testicular interstitium in two very different physiological situations involving testicular involution: ageing and exposure to a short photoperiod. The animals were divided into an 'age group' with three subgroups - young, adult and old animals - and a 'regressed group' with animals subjected to a short photoperiod. The testicular interstitium was characterised by light and electron microscopy. Interstitial cells were studied histochemically with regard to their proliferation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling (TUNEL+) and testosterone synthetic activity. We identified two types of Leydig cell: Type A cells showed a normal morphology, while Type B cells appeared necrotic. With ageing, pericyte proliferation decreased but there was no variation in the index of TUNEL-positive Leydig cells. In the regressed group, pericyte proliferation was greater and TUNEL-positive cells were not observed in the interstitium. The testicular interstitium suffered few ultrastructural changes during ageing and necrotic Leydig cells were observed. In contrast, an ultrastructural involution of Leydig cells with no necrosis was observed in the regressed group. In conclusion, the testicular interstitium of Mesocricetus auratus showed different cellular changes in the two groups (age and regressed), probably due to the irreversible nature of ageing and the reversible character of changes induced by short photoperiod.
    Reproduction Fertility and Development 12/2014;
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    ABSTRACT: Studies have shown that gonadotropin-releasing hormone-based protocols that reduce the period of progestin insertion and prolong the period from progestin removal to gonadotropin-releasing hormone and fixed-time AI (FTAI; named 5-day Co-Synch) results in similar or higher pregnancy rates than the conventional 7-day Co-Synch protocol in beef cows and heifers (Bridges et al. 2008 Theriogenology 69, 843-851). Similar findings have been reported following the use of an oestradiol-based protocol that also provides for a longer period of proestrus (named J-Synch; de la Mata and Bó 2012 Taurus 55, 17-23). An experiment was designed to compare the J-Synch protocol for synchronization of ovulation that allows for a prolonged proestrus with a conventional 7-day oestradiol-based protocol for FTAI in heifers. Cycling 18-month old Angus and Hereford heifers (n=208) with a body condition score of 6 to 7 (scale of 1 to 9) were randomly allocated to 1 of 2 treatment groups. Heifers in the 7-day EB group (n=105) received a progesterone (P4) device (DIB 0.5g of P4, Syntex SA, Buenos Aires, Argentina) and 2mg of oestradiol benzoate (EB, Syntex SA) on Day 0 and 500μg of cloprostenol (PGF; Ciclase DL, Syntex SA) and 0.5mg oestradiol cypionate (Cipiosyn, Syntex SA) on the day of DIB removal (Day 7). Heifers were also tail painted at the time of DIB removal and observed for signs of oestrus (i.e. tail paint rubbed off). Those with the tail paint rubbed off by 36h after DIB removal were inseminated 12h later, whereas those not showing oestrus by 36h were FTAI at 54h. Heifers in the J-Synch group (n=103) received DIB and 2mg of EB on Day 0 and PGF on the day of DIB removal (Day 6). Heifers in this group were also tail painted at DIB removal, and those with their tail paint rubbed off by 48h were inseminated 12h later; those not showing oestrus by 60h received 100μg of gonadorelin acetate (gonadotropin-releasing hormone, Gonasyn gdr, Syntex SA) and were FTAI at 72h after DIB removal. Pregnancy was diagnosed by ultrasonography at 55 days after FTAI (Honda 101V, 5.0-MHz transducer). Data were analysed by logistic regression. Oestrus detection rate and pregnancy rate to FTAI did not differ (P>0.1) between groups (38.8%, 40/103 and 60.3%, 38/ 63 for heifers in the J-Synch group v. 28.5%, 30/105 and 45.3%, 34/75 for those in the 7-day EB group). However, pregnancy rates to observed oestrus tended (P<0.09) to be higher and the overall pregnancy rate was significantly higher (P<0.01) in heifers in the J-Synch group (80.0%, 32/40 and 67.9%, 70 /103) compared with those in 7-day EB group (50%, 15/30 and 46.6%, 49/105). Furthermore, heifers within the J-Synch group that had their tail paint rubbed off by 48h after DIB removal and were AI 12h later (i.e. 60h) had higher (P<0.05) pregnancy rate than those in the same group that were FTAI. In conclusion, reducing the time of progestin device insertion and lengthening the proestrus period, as in the J-Synch protocol, results in higher pregnancy rates than with the conventional oestradiol-based protocol. Furthermore, the combination of oestrus detection and FTAI would appear to improve the pregnancy outcome even more.
    Reproduction Fertility and Development 12/2014; 27(1):96-7.
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    ABSTRACT: Previous studies have demonstrated that a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillmaine, SNAP) and a phosphodiesterase inhibitor (Sildenafil, SILD) delay the meiotic resumption of oocytes removed from the follicular environment, and therefore could be used to improve the quality of in vitro-matured (IVM) oocytes. However, it has been reported that SILD-treated cells have increased lipid metabolism and that NO supplementation can modulate the oxidative stress. This study aims to determine the effects of SNAP or SILD supplementation, or both, during IVM on embryo developmental rates, on lipid accumulation of IVM oocytes and on reactive oxygen species (ROS) and lipid accumulation of embryos derived from IVM oocytes. Bovine oocytes were cultured in TCM199 containing 1.0μgmL(-1) of FSH, 50μgmL(-1) of hCG, 1.0μgmL(-1) of oestradiol, 0.2mM pyruvate, 83.4μgmL(-1) of amikacin, 10% FBS (control group; GCONT), supplemented with 10µM SILD (GSILD), 0.1µM SNAP (GSNAP) or both (GS+S). After 24h of IVM, matured oocytes were assessed for lipid quantification (approximately 49 per group) or used for in vitro embryo production (IVP; approximately 340 oocytes per group). For lipid quantification, denuded oocytes were fixed with 5% triton in 4% paraformaldehyde (PFA) for 30min and stained with 1ngmL(-1) of Nile Red for 30min. Embryo lipid analyses (approximately 55 per group) were performed as described for oocytes. For ROS assessment (approximately 58 per group), IVP embryos were stained with 10µM of H2DFFDA for 1h and fixed for 30min in 4% PFA. Stained oocyte and embryo assessments were performed on epifluorescence microscopy, and captured images were analysed on ImageJ (NIH, Bethesda, MD, USA) to quantify the fluorescence intensity (f.i). Statistical analyses were performed with data from 3 replicates for oocytes and 4 for embryos: statistical differences were assessed for lipid and ROS quantity and development rates by split-plot ANOVA. Variables considered in the model were SNAP (presence/absence) and SILD (presence/absence). Means were compared by Student's t at P<0.05. Regarding oocyte lipid accumulation, groups with SILD (GSILD and GS+S) presented higher lipid quantity (f.i: 52.11 and 47.24, respectively) compared with GCONT and GSNAP (f.i: 38.86 and 41.86, respectively). Supplementation during IVM did not affect development rates (cleavage of 88.1, 88.2, 88.8, and 89.5% and blastocyst rates of 41.2, 38.6, 40, and 41.2% for GCONT, GSNAP, GSILD, and GS+S, respectively). Regarding embryo lipid quantity, similar to oocyte results, SILD groups (GSILD and GS+S) presented higher lipid accumulation (f.i: 68.9 and 68.5, respectively) compared with GCONT (f.i: 55.8) and GSNAP (f.i: 58.9). Considering embryo ROS quantity, GCONT (f.i: 35.9) and GS+S (f.i: 34.2) had the highest levels; however, GS+S did not differ from GSNAP (f.i: 32.85), which was similar to GSILD (f.i: 30.4). In conclusion, SILD had a negative effect on lipid accumulation, which could be due to increased lipid synthesis without increasing lipid oxidation because no increase of embryo ROS levels was observed.
    Reproduction Fertility and Development 12/2014; 27(1):213.
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    ABSTRACT: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) technology is considered as an efficient strategy for generating gene edited large animals, such as pigs. Compared to somatic cell nuclear transfer, this new technology offers a relatively simple way to generate mutant pigs by direct injection of RNA into the cytoplasm of zygotes. Moreover, the use of in vitro produced zygotes would provide a highly effective and practical method for the production of porcine disease models for biomedical research. Here we examined the production efficiency of growth hormone receptor (GHR) mutant pigs by the combination of the CRISPR/Cas system and in vitro produced zygotes. In vitro maturation (IVM) of oocytes was performed as described previously (Kurome et al., Meth. Mol. Biol., in press). In all experiments, the same batch of frozen sperm was used. After IVM, around 20 oocytes with expanded cumulus cells were incubated with 5×10(4) spermatozoa in a 100-μL drop of porcine fertilization medium for 7h. In vitro-produced embryos were assessed by the ratio of normal fertilization (eggs with 2 pronuclei) and blastocyst formation at Day 7. The Cas9 mRNA and a single guide RNA, recognising a short sequence of 20 base pairs in exon 3 of the GHR gene, were injected directly into the cytoplasm of the embryos 8.5 to 9.5h after IVF. Injected embryos were transferred laparoscopically to recipient pigs, and 86.4% (57/66) of sperm-penetrated oocytes (66/96) exhibited normal fertilization. Incidence of polyspermy was relatively low (9/66, 13.6%). Developmental ability of in vitro-produced embryos to the blastocyst stage was 17.4% (24/138). In total, 426 RNA-injected embryos were transferred into 2 recipients, one of which became pregnant and gave birth to 8 piglets. All piglets were clinically healthy and developed normally. In 3 out of 8 piglets (37.5%), mutations were introduced. Next-generation sequencing revealed that all of them were mosaics: one with a single mutation (22% wild-type/78% mutant) and 2 piglets with 2 different mutations (80% wild-type/2% mutant_1/18% mutant_2 and 94% wild-type/4% mutant_1/2% mutant_2). Four out of 5 mutations caused a frameshift in the GHR gene. Our study reports for the first time generation of GHR mutant pigs by the use of the CRISPR/Cas system in in vitro-produced zygotes. Because all GHR mutant offspring were mosaic, Cas9 activation probably occurred after the 1-cell stage under our experimental conditions. The founder animal with the highest proportion of mutant GHR alleles will be used for breeding to establish a large animal model for Laron syndrome.
    Reproduction Fertility and Development 12/2014; 27(1):269.
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    ABSTRACT: Immature equine oocytes may be held overnight in an Earle's/Hanks' M199-based medium in the absence of meiotic inhibitors (EH medium) to schedule the onset of in vitro maturation. Holding in EH has been shown not to affect meiotic or developmental competence of equine oocytes (Choi et al. 2006 Theriogenology 66, 955-963). However, no studies have been performed to identify the mode by which this medium suppresses meiosis. We hypothesised that holding temperature may affect oocyte meiotic arrest. The effect of 3 holding temperatures (25, 30, 38°C) on chromatin status was investigated after Hoechst 33258 staining (Hinrichs et al. 2005 Biol. Reprod. 72, 1142-1150). Oocytes were recovered by scraping of follicles from slaughterhouse-derived ovaries. Data were analysed by Chi-squared test and one-way ANOVA followed by Dunn's or Holm-Sidak Multiple Comparison methods. A level of P<0.05 was considered significant. There were no significant differences in chromatin configuration between oocytes held overnight at 25°C (25°C-held) and controls (immediately-fixed oocytes); the proportion of oocytes showing meiotic resumption was 1/27, 4% and 0/26, 0%, respectively (not significant, NS). In contrast, holding at higher temperature significantly increased meiosis resumption (14/38, 37% and 14/28, 50%, at 30 and 38°C, respectively; P<0.01) and reduced the proportion of oocytes showing the most meiotically-competent germinal-vesicle (GV) configuration (condensed chromatin, CC; 24 to 29% v. 65 to 70% for control and 25°C-held, respectively; P<0.05). Based on these results, a subsequent experiment was performed in which oocyte meiotic stage and mitochondrial (mt) potential of 25°C-held (n=29) and control (n=36) oocytes was evaluated. Nuclear chromatin, mt activity (MitoTracker orange), intracellular reactive oxygen species (ROS) levels (2',7'-dichlorodihydrofluorescein diacetate, DCDHFDA), and mt/ROS colocalization (Pearson's coefficient) were analysed by epifluoscence and confocal microscopy (Martino et al. 2012 Fertil. Steril. 97, 720-728). Meiotic arrest after EH treatment at 25°C was confirmed (0/29, 0% v. 5/36, 14% for meiotic resumption in 25°C-held and controls, respectively; NS). At any GV stage, 25°C-held treatment had no effect on mt activity, ROS levels, or mt/ROS colocalization. For example, in CC oocytes, values for control and 25°C-held, respectively, were: MitoTracker, 547.8±499.5 v. 722.9±390.3; DCF fluorescence intensity, 278.5±179.3 v. 378±185, and mt/ROS colocalization, 0.5±0.1 v. 0.5±0.2; these were not significantly different (NS). In conclusion, EH holding at 25°C maintains meiotic arrest, viability, and mt potential of equine oocytes.
    Reproduction Fertility and Development 12/2014; 27(1):244.
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    ABSTRACT: The multiplication of high-value embryos by chimera formation using asynchronic aggregation is a promising alternative to embryonic cell nuclear transfer. Single blastomeres from a donor embryo are aggregated with 2 host embryos, thus several chimeras can be constructed per donor embryo. Due to the advanced developmental stage, the donor blastomeres are likely to contribute to the inner cell mass (ICM) and later give rise to the embryo proper, whereas the host embryos form extra-embryonic tissues. To test if pairs of blastomeres from Day 5 morulae are able to form the ICM when aggregated with 2 Day 4 host embryos, we produced transgenic donor embryos carrying a fluorescent reporter gene (enhanced green fluorescent protein, eGFP) by using semen from an eGFP transgenic bull (Reichenbach et al. 2010 Transgenic Res. 19, 549-556) for in vitro fertilization and in vitro host embryos produced by a standard procedure. The zona pellucida of all embryos was removed by treatment with 1mgmL(-1) pronase. Donor embryos were assessed for eGFP expression by fluorescence microscopy and disaggregated by gentle pipetting after incubation in Mg(2+)- and Ca(2+)-free medium. Pairs of blastomeres were then placed between 2 host embryos and cultured individually in a well-of-the-well culture dish. On Day 6 after aggregation, fully developed blastocysts were assessed for eGFP fluorescence. In 3 replicates, n=30 chimeras were produced by aggregation; 13 (43%) developed to blastocysts, of which 2 (15%) showed local eGFP expression in the ICM and 7 (54%) showed a generalized expression. From the results of this study we conclude that Day 5 morulae may be multiplied in an efficient manner by using the chimera formation technique, which makes this approach applicable to ex vivo-derived embryos. In future investigations we will study the effect of using donor blastomeres from either the inside or outside of the donor morula and test the use of tetraploid host embryos to increase the rate of blastocysts with the desired genotype in the ICM. Finally, we aim to introduce this multiplication approach to the production of genotyped embryos with a genomic estimated breeding value (gEBV) and intend to produce calves with identical gEBV.
    Reproduction Fertility and Development 12/2014; 27(1):135.
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    ABSTRACT: The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species. We propose that in vitro sperm capacitation could optimize the ICSI in cattle. The aim was to evaluate the effects of isobutylmethylxanthine (IBMX) and methyl-β-cyclodextrin (MβCD) on the sperm capacitation and in vitro development of embryos generated by ICSI. Frozen-thawed spermatozoa (3-5×10(6) cellsmL(-1)) were pre-incubated for 2h at 38.5°C, 5% CO2 in defined medium (Sp-TLP/PVA) supplemented with MβCD (1mM) or IBMX (0.4mM) (capacitating conditions). The untreated control group (UTG; not supplemented) and vehicle group (VG) were incubated for 2h. The non-capacitating control group (NCG) was not supplemented (neither vehicle nor IBMX or MβCD) and not incubated. The sperm viability and capacitation {intracellular calcium [Ca(2+)]i, plasma membrane fluidity (PMF), and acrosomal reaction} were evaluated by flow cytometry (n=3 biological replicates). For the ICSI procedure, only motile spermatozoa were selected. After ICSI, oocytes were activated with ionomycin+cycloheximide. Culture was performed at 38.5°C, 5% CO2, 5% O2, 90% N2, saturation humidity in KSOM base medium. Data were analysed by ANOVA and Scheffe's test. Pronuclear formation was evaluated by a chi-square test with Bonferroni's correction. Significance was set at P<0.05. Pretreated spermatozoa showed lower (P<0.05) viability (49 and 67% for IBMX and MβCD, respectively) compared with the NCG (89%), UTG (80%), and VG (78%). The [Ca(2+)]I analysed by median fluorescence intensity (MFI) was lower (P<0.05) in NCG (117 MFI) with respect to UTG (127 MFI), VG (124 MFI), IBMX (126 MFI), and MβCD (131 MFI). The PMF increased (P<0.05) with IBMX (115 MFI) and MβCD (106 MFI) compared with NCG (70 MFI), UTG (89 MFI), and VG (65 MFI). Acrosome reaction improved with capacitating treatments with respect to both control groups (16, 23, 8, 4, and 3% for IBMX, MβCD, UTG, VG, and NCG, respectively). Analysis of capacitating v. non-capacitating conditions on ICSI efficiency revealed that the fertilization rate, assessed by pronuclear formation, was higher (P<0.05) in ICSI-MβCD (76%; n=46) compared with ICSI-IBMX (55%; n=53) and ICSI-NCG (50%; n=44). Nevertheless, there were no differences among groups in cleavage (Day 3): 85, 86, and 84% and blastocyst rates (Day 8): 19, 25, and 18% for ICSI-IBMX (n=8), ICSI-MβCD (n=7), and ICSI-NCG (n=7), respectively. The parthenogenetic and sham injection groups yielded a lower rate of cleavage (73 and 53%, respectively) and blastocyst (13% and 10%, respectively). The results demonstrated an improvement of the fertilization rate of bovine embryos generated by ICSI using sperm capacitated by MβCD pretreatment. However, more studies are necessary to improve in vitro developmental potential of these embryos to the blastocyst stage.
    Reproduction Fertility and Development 12/2014; 27(1):248.
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    ABSTRACT: Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, is expressed in human and mice pre-implantation embryos and reproductive tract (Li et al. 2009 FEBS Lett. 583; Kawamura et al. 2012 PLoS One 7). In mice, artemin promotes in vitro embryo development and decreases apoptosis (Li et al. 2009 FEBS Lett. 583). The presence of artemin in cattle embryos and endometrium, however, is unknown. In this work we analysed artemin expression in bovine blastocysts and endometrium by immunohistochemistry and in uterine fluid (UF) by Western blot (WB). Briefly, Day-6 in vitro-produced (IVP) embryos (n=50) were nonsurgically transferred to the uterus of heifers (n=10, 50 IVP embryos per heifer) at nonconsecutive oestrus cycles. On Day 8, embryos and their corresponding diluted UF were flushed; blastocysts that developed entirely in vitro were also collected. In addition, endometrial samples were collected on Day 8 from slaughtered females that were embryo transferred (n=6) and sham transferred (n = 6) on Day 5. Artemin localization was investigated in blastocysts and in endometrial samples, using immunohistochemical staining methods described elsewhere (Muñoz et al. 2012 J. Proteome Res. 11; Gómez et al. 2014 Reproduction pii: REP-14-0304). The signal-strength comparisons between uterus-exposed and IVP blastocysts were analysed using the software Confocal Uniovi Image-J. Quantification of WB protein bands was achieved by computer-assisted densitometry using Image-J software. Artemin was detected, with similar intensity, in the inner cell mass and trophectoderm from both uterus-developed and IVP blastocyst. All embryos analysed expressed artemin. The signal intensity and staining pattern observed did not differ between uterus-exposed and IVP blastocysts. In the endometrium, the most intense staining for artemin was localised to the apical sites in the luminal epithelium and in the glandular epithelium of superficial glands. There was also diffuse staining in the stroma and deep uterine glands. The uterine region and pregnant or cyclic status did not affect the artemin staining pattern. Artemin was detected by WB in all UF samples analysed (embryo transferred N=10, sham transferred N=10). However reliable quantitation of artemin by WB was unfeasible due to the broad dynamic range of artemin expression through samples. In conclusion, our results demonstrate the presence of artemin in bovine uterine endometrium and UF, and embryos during early development. As shown in mice, it is feasible that artemin might exert an autocrine/paracrine role during early embryo development in the cow.
    Reproduction Fertility and Development 12/2014; 27(1):165-6.
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    ABSTRACT: The generation of transgenic cell lines through standard cell transfection/antibiotic selection procedures may have a negative effect on cell viability, which in turn may compromise SCNT cloning efficiency. The aim of this study was to evaluate goat cloning efficiency by using transfected and nontransfected and transgenic and nontransgenic somatic cells as nucleus donors. Skin fibroblast cells from 1 adult doe were subjected to transfection by electroporation with the pBC1-hGCase-Neo transgene cassette containing the human glucocerebrosidase gene sequence (hGCase), following antibiotic cell colony selection. Four distinct syngeneic donor cell types were used for cloning: (a) wild type (nontransfected, nontransgenic) control cells (C1) at low passage (P3), (b) transfected negative control (transfected, nontransgenic) cells (CT) at high passage (P8), and (c) 2 lines of transfected, transgenic cells (CA, CB) at high passages (P8 through P10). Donor cell cycles were synchronized by high confluence (<95%) and 24-h serum starvation. Cloning procedures were performed by standard micromanipulation procedures. Following membrane fusion after a 1.25kVcm(-1) DC pulse for 45µs, reconstructed structures were incubated in cytochalasin B for 1h, and then activated in ionomycin/6-DMAP. After 12h of IVC in G-1(TM) medium (Vitrolife, Englewood, CO, USA), 1-cell stage cloned embryos were surgically transferred into the oviduct of synchronous recipient females. To ascertain herd fertility and health and adequate procedures for embryo manipulation, synchronization protocols, and surgical interventions, groups of control females were subjected to cervical AI or surgical transfer of in vivo-produced 1-cell stage goat embryos (ET). Pregnancy diagnosis was performed by ultrasonography on Day 23, with weekly examinations until term. Data were analysed by the χ(2) test (P<0.05), and are presented in Table 1. The transfection process and passage number did not appear to affect development, as no differences in pregnancy rates were observed between cloned groups, although results with control cells (C1 and CT) and with CA and CB lines were similar to and lower than the AI and ET groups, respectively. Loss rate after cloning was high (88.8%), which may be due to faulty reprogramming, as other procedural and biological variables involved in the cloning process were endorsed by pregnancy rates and term viable pregnancies observed in the AI and ET groups. Cloning using CA donor cells at P9 resulted in two liveborn kids, with one dying soon after birth. Both animals were confirmed by molecular analyses as hGCase transgenic clones.
    Reproduction Fertility and Development 12/2014; 27(1):111.
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    ABSTRACT: Prepubertal bovine donors are currently used for commercial breeding to accelerate the genetic gain and decrease the generation interval. Nevertheless, it has been reported that their oocyte developmental competence is lower than in adult females. Addition of cAMP regulators during in vitro maturation (IVM) has been suggested to enhance blastocysts rates (Albuz et al. 2010 Hum. Reprod. 25, 2999-3011). Here, we evaluated the effects of the cAMP modulators forskolin, 3-Isobutyl-1-methylxanthine (IBMX), and cilostamide during extended IVM on the developmental capacity of oocytes from prepubertal and adult bovine females. A total of 1851 oocytes from 24 lactating cows (>2 lactations) and 24 prepubertal donors (6-10 mo old) were collected by transvaginal oocyte recovery (OPU) twice per week and divided into 3 experiment groups: (1) TCM24 (OPU medium: PBS; 24h of IVM; standard protocol/control); (2) cAMP30 [OPU medium: PBS-IBMX (500μM); 2h pre-IVM culture using forskolin (100μM)-IBMX (500μM) and 30h of IVM adding cilostamide (20μM)], and (3) DMSO30 [cAMP modulators are diluted in DMSO)/vehicle control; OPU medium: PBS-DMSO (46.3mM); 2h pre-IVM culture (280mM DMSO) and 30h IVM (5.6mM DMSO)]. Following IVM, oocytes were either submitted to in vitro fertilization and embryo culture or fixed in 1% glutaraldehyde at 9, 20, 24, and 30h after IVM and stained with Hoechst to evaluate their nuclear status. One-way ANOVA was implemented to evaluate recovered oocytes and meiotic stages. The Glimmix procedure from SAS/STAT was performed to compare blastocyst and cleavage rates. Total number of oocytes and IVM-suitable oocytes per donor per OPU session were similar in adult and prepubertal donors (total number/IVM suitable; prepubertal donors: 6.7/4.2, 6.4/4.0, 6.5/3.8; cows: 6.2/4.7, 6.2/4.4, 6.2/4.5 for TCM24, cAMP30 and DMSO30, respectively). At 9h, cAMP regulators were able to maintain meiotic arrest in prepubertal and adult donors (GV: 80.0 and 40.9%, respectively) compared to standard IVM (GV: 61.1 and 31.2%) and DMSO30 (GV: 40.0 and 26.6%) protocols (P<0.05). Using the cAMP30 protocol, the percentage of oocytes that reached MII stage at 20h was lower in adult (4.5%) and prepubertal donors (5.26%) compared to the DMSO30 (50.0 and 42.8%, respectively) and TCM24 (56.2 and 44.4% respectively) protocols. Metaphase II rates after either 24 or 30h were similar among treatments (prepubertal donors: 88.2, 70.5, and 84.2%; cows: 71.4, 85.7, and 81.2% for TCM24, cAMP30, and DMSO30, respectively; P>0.05). Cleavage rates (prepubertal donors: 63.4, 54.9, and 52.1%, cows: 56.1, 57.8, and 51.6% for TCM24, cAMP30, and DMSO30, respectively) and blastocysts/presumptive zygotes rates (prepubertal donors: 26.2, 19.6, and 16.2%; cows: 27.5, 28.1, and 21.5% for TCM24, cAMP30, and DMSO30, respectively) did not show significant differences (P>0.05). Although cAMP modulators delayed the progression through meiosis in adult and prepubertal oocytes, similar blastocysts rates were obtained. Our results suggest so far that oocyte retrieval and competence in prepubertal donors can be similar to that of the adult donors with and without addition of cAMP modulators.
    Reproduction Fertility and Development 12/2014; 27(1):232.
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    ABSTRACT: A major problem of embryos cultured in vitro with serum is cytoplasmic lipid accumulation resulting in lower cryotolerance compared with those derived from in vivo or in the absence of serum. AMPK is known as a master regulator of lipid, glucose, and protein metabolism in mammalian cells. Moreover, it has been reported as controller of acetyl-CoA carboxylase α (ACC), the gene responsible for lipid synthesis, and associated with mitochondrial biogenesis and activities in response to oxidative stress. In the present study we aimed to investigate the regulation of AMPK during serum supplementation in vitro. For this, bovine embryos were produced in vitro in SOF media supplemented with oestrous cow serum or fatty acid-free BSA as a system without serum. Triplicate pools (each 10 blastocysts) from each group were used for RNA isolation using Arcturus(®)PicoPure(®)RNA Isolation Kit (Life Technologies, USA). Reverse transcription was performed using a combination of Oligo(dT)23 and random primers. Quantification of AMPK catalytic α1 (AMPKA1), ACC, peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1A), and sterol regulatory element binding transcription factor 2 (SREBP2) transcripts were performed using ABI PRISM(®) 7000 SDS system (Applied Biosystems, Foster City, CA, USA) using GAPDH as internal control. Normalized log-transformed transcript amount data were statistically analysed using t-test. In addition, AMPK protein was detected by immunofluorescence, mitochondrial activity by MitoTracker(®) Red (Invitrogen, Carlsbad, CA, USA), and reactive oxygen species by H2DCFDA molecular probe (Life Technologies, USA), and fluorescent intensity signals were visualised under confocal laser scanning microscopy LSM 710 (Carl Zeiss, Germany). Results showed that the expression of AMPKA1, PGC1A, a mitochondrial biogenesis protein, and SREBP2, a regulator of lipid oxidation, were found to be lower (0.4-, 0.2-, and 0.7-fold, respectively; P<0.05) in blastocysts derived from cultured with serum compared to without serum. By contrast, ACC was up-regulated in blastocysts cultured with serum by 1.8-fold (P<0.05) compared to without serum. In comparison to blastocyst cultured without serum, a reduced fluorescent intensity was observed in AMPKA1 protein and mitochondrial activity in blastocyst cultured with serum. The presence of serum was also found to be involved in increasing reactive oxygen species accumulation in embryos cultured with serum. The reduced level of AMPK leads to increased ACC and subsequently enhanced conversion of fatty acids into lipid, which is associated with reduced mitochondrial biogenesis protein, elevated reactive oxygen species level, and reduced lipid oxidation by suppression of SREBP2. In conclusion, the presence of serum in in vitro culture environment affected the AMPK activity and thereby genes associated with lipid metabolism in early bovine embryos.
    Reproduction Fertility and Development 12/2014; 27(1):156.