- Fibrin–alginate hydrogel supports steroidogenesis, in vitro maturation
of oocytes and parthenotes production from caprine preantral follicles
cultured in group
- Abstract: This study aimed to establish a culture system that improves the in vitro development of caprine preantral follicles. In a first experiment, follicles were encapsulated as a single unit per bead and cultured singly or in groups or with five follicles in the same alginate (ALG) bead for 18 days. In a subsequent experiment, the “five follicles per bead” design was chosen to culture in ALG, fibrin–alginate (FA) or hyaluronate (HA) for 18 days. In a third experiment, we chose the five follicles per bead in FA to culture for 30 days. The culture set‐up of five follicles per ALG bead increased antrum formation and follicle diameter compared to the other culture designs (p
- Molecular expression of caprine estrogen receptor gene 1 in reproductive
and non‐reproductive tissues
- Authors: S Saraswat; PK Rout, SD Kharche, SK Jindal, AK Goel
Abstract: During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high‐fertile (G1) and low‐fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real‐time PCR (Roche LC‐480). Our work shows that the relative quantification by RT‐PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT‐PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.
- Composition of seminal plasma and ovarian fluid in Ide Leuciscus idus and
Northern pike Esox lucius
- Authors: MAM Siddique; O Linhart, R Kujawa, S Krejszeff, IAE Butts
Abstract: Seminal plasma (SP) and ovarian fluid (OF) plays an important role as storage media to prevent the activation of gametes both in vivo and under artificial conditions. The objectives of this study were to quantify gamete biochemistry and explore correlations among quantitative characteristics of SP, OF and sperm performance traits of Ide Leuciscus idus and Northern pike Esox lucius. Generally, Na+, K+ and Cl− were found to be the most dominating ions, although concentrations of K+ were higher in SP, while Na+ and Cl− concentrations were higher in OF for both species. Several significant correlations among the biochemical properties such as total protein, glucose, osmolality, cholesterol, K+, Ca2+, Cl− and Mg2+ were observed for SP and OF. Total protein content of Ide SP was positively correlated with sperm activity traits (r ≥ .89, p ≤ .05), while K+ concentration was negatively correlated with sperm traits (r ≥ −.89, p ≤ .05). Moreover, Ca2+ concentration in Northern pike SP was positively correlated with the percentage of sperm motility (r = . 98, p
- Beta‐mercaptoethanol supplementation of in vitro maturation medium does
not influence nuclear and cytoplasmic maturation of equine oocytes
- Authors: B Merlo; E Iacono, D Bucci, M Spinaci, G Galeati, G Mari
Abstract: 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.
- Cumulus cell expansion and first polar body extrusion during in vitro
oocyte maturation in relation to morphological and morphometric
characteristics of the dromedary camel ovary
- Authors: F Mesbah; M Kafi, H Nili
Abstract: The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6‐hr interval) in Hams‐F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4‐year‐old and >4‐year‐old dromedary camel (p 4‐year‐old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2–6 mm follicles are cultivable. The most slaughterhouse‐derived COCs retrieved from 2–6 mm follicles of non‐pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24–30 hr; the optimal time for CC expansion is 24–30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM.
- Semen characteristics of rainbow trout (Oncorhynchus mykiss) following
diets containing different vegetable fatty acid levels
- Authors: M Hajiahmadian; K Sarvi Moghanlou, N Agh, F Farrokhi Ardabili
Abstract: Brood fish nutrition is an important factor susceptible to affect not only fecundity and gametogenesis but also gamete quality. In this study, we investigated the effects of altering dietary vegetable fatty acid content on semen quality (i.e. motility, density and seminal plasma composition), fertilizing ability and also blood testosterone (T) concentration in rainbow trout (Oncorhynchus mykiss). Fish were fed a commercial diet and ten formulated diets with similar proximate compositions but different levels of vegetable fatty acids (highly unsaturated fatty acids (HUFA): monounsaturated fatty acids (MUFA); HUFA: polyunsaturated fatty acids (PUFA); and HUFA: saturated fatty acids (SFA) ratios). Fish fed with HUFA: MUFA = 0.0 and HUFA: SFA = 0.25 ratios had the highest semen motility percentage and duration. However, the highest semen concentration and semenatocrit were observed in HUFA: SFA = 0.0 and HUFA: PUFA = 0.37 ratios. There was a significant difference in terms of K+ ion among diets supplemented with HUFA: PUFA = 0.0, HUFA: PUFA = 0.37 and HUFA: MUFA = 0.16 ratios (p
- Reduction in the mRNA expression of sVEGFR1 and sVEGFR2 is associated with
the selection of dominant follicle in cows
- Abstract: The vascular endothelial growth factor (VEGF) is essential for follicular development by promoting follicular angiogenesis, as well as for the proliferation and survival of granulosa cells. The biological effects of VEGF are regulated by two membrane receptors, VEGFR1 and VEGFR2, and two soluble receptors, sVEGFR1 and sVEGFR2, which play an antagonistic role. Thus, the objective of this study was to identify the mRNA expression pattern of total VEGF, VEGFR1, VEGFR2, sVEGFR1 and sVEGFR2 in bovine preselected follicles (PRF) and post‐selected follicles (POF). The mRNA expression of these five genes in both granulosa cells (GC) and theca cells (TC) was compared between follicles classified as PRF and POF based on their diameter and on their ratio of estradiol/progesterone (E2/P4). Results showed a lower expression of mRNA of sVEGFR1 and sVEGFR2 in POF than in PRF (p .05). Our results showed that the mRNA expression of VEGFR2 and sVEGFR1 was more abundant than the expression of VEGFR1 and sVEGFR2, while GC was the main source of mRNA for total VEGF. On the other hand, TC was the follicular compartment where the receptors were most expressed. Our results suggest that non‐dominant follicles maintain a greater concentration of the mRNA expression of both membrane and soluble VEGF receptors. On the other hand, follicular dominance is related to a reduction in the mRNA expression of sVEGFR1 and sVEGFR2, which may favour VEGF binding with VEGFR2 and, hence, improve the follicular health and development.
- Lipid profiles of canine spermatozoa as revealed via matrix‐assisted
laser desorption/ionization mass spectrometry
- Authors: LT Braga; NRS Bravo, KRA Belaz, D Zampieri, MN Eberlin, VA Conforti
Abstract: In this study, we investigated the ability of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) to characterize the lipid contents of canine spermatozoa. For that, samples of pure semen were analysed. Indeed, quite comprehensive lipid coverage was observed, and the most abundant phospholipid ions detected were from four phosphatidylcholines, that is those of m/z 760.6; 782.6; 808.6; and 830.6 and one of m/z 725.6 from a sphingomyelin. In conclusion, MALDI‐MS was found to offer an easy, fast, accurate, and sensitive analytical method for lipid profiling in canine spermatozoa and could be used as a tool to select sires by assessing the relationship between sperm lipid profiles and variables such as age and breeding history as well as to study the effects of cryopreservation on lipid contents.
- Transcriptome analysis of the uniparous and multiparous goats ovaries
- Authors: LJ Wang; XW Sun, FY Guo, YJ Zhao, ZH Zhang, ZQ Zhao
Abstract: Transcriptome analysis of Inner Mongolia Cashmere goat and Dazu black goat generated 38,772,947 and 38,771,668 clean pair end reads, respectively, which were assembled into 72,422 and 80,069 unigenes by Trinity, respectively. For Inner Mongolia Cashmere goat, 26,051 and 10,100 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups, respectively. A total of 32,772 unigenes can comment to SWISS‐Prot database, and the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 24,420 unigenes. While annotating the unigenes about Dazu black goats, we found 29,444(45.42%), 11,271 (38.28%), 36,910(56.94%) and 27,766 (42.83%) unigenes were assigned to GO database, COG database, SWISS‐Prot database and KEGG database, respectively. In addition, we performed the bioinformatics analysis of gene expression profiling aimed at the ovarian transcriptome difference between Inner Mongolia Cashmere goat and Dazu black goat. We obtained a sequencing depth of over 5.5 million and 5.8 million tags. There were 1,133 DEGs between two species, of which 632 genes upregulated in the Dazu black goat and 501 genes downregulated compared with which in Inner Mongolia Cashmere goat. By annotating the 1,133 DEGs into KEGG database, we found 525 DEGs. And there were 68 DEGs annotated in metabolic pathways, 31 DEGs annotated in ribosome, 28 DEGs annotated in focal adhesion, 27 DEGs annotated in phagosome, 26 DEGs annotated in pathways in cancer, 25 DEGs annotated in ECM‐receptor interaction, 23 DEGs annotated in protein digestion and absorption, 20 DEGs annotated in oxidative phosphorylation, 17 DEGs annotated in lysosome, and 16 DEGs annotated in cell adhesion molecules.
- The effects of canthaxanthin on porcine oocyte maturation and embryo
development in vitro after parthenogenetic activation and somatic cell
- Authors: A Taweechaipaisankul; JX Jin, S Lee, GA Kim, BC Lee
Abstract: The objective of this study was to examine the effects of canthaxanthin (Cx) treatment during in vitro maturation (IVM) of porcine oocytes on embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), on intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in mature oocytes, and on gene expression in both PA‐ and SCNT‐derived blastocysts. To determine the optimal effective concentration of Cx, porcine oocytes were cultured in IVM medium supplemented with various concentrations (0, 20, 40 and 80 μM) of Cx for 22 hr. Compared to other groups, supplementation with 40 μM Cx significantly improved blastocyst formation rates after PA (p
- Lysophosphatidic acid expression in theca cells depends on the type of
bovine ovarian follicle
- Abstract: Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte‐cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1‐4) were determined. The obtained results confirmed the follicle‐type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1‐4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action.
- Effect of short‐term exposure of cumulus–oocyte complex to
3‐morpholinosydnonimine on in vitro embryo development and gene
expression in cattle
- Abstract: Short‐term exposure of gametes to different types of stress might induce stress tolerance in mammalian embryos. The aim of this study was to evaluate the effect of short‐term exposure of bovine mature cumulus–oocyte complex (COC) to 3‐morpholinosydnonimine (SIN‐1) on subsequent in vitro embryo development, embryo quality and relative gene expression. Matured COCs were incubated with SIN‐1 (0, 0.1, 1, 10 and 100 μM SIN‐1) for 1 hr before in vitro fertilization and zygotes were cultured until Day 7. The cleavage rate at 72 hr did not show any differences among groups. However, the blastocyst rate on Day 7 decreased with all treatments evaluated, with the embryos generated with 10 μM SIN‐1 showing the lowest embryo production rate. Embryo quality analysis did not show any differences in total cell number (TCN) or inner cell mass (ICM) among groups. Relative gene expression analysis showed a downregulation of eNOS expression and an upregulation of nNOS expression in all treatments evaluated compared to the control group. Also, a downregulation was observed in some treatments: SOD2 at 0.1 μM; SOD1 at 0.1 and 100 μM; PRDX5 at 0.1, 10 and 100 μM; and NANOG at 10 and 100 μM; and an upregulation of CDX2 expression was observed at 100 μM. The other genes (OCT4, HIF1A, HSPA1A, BCL2A and iNOS) did not show any differences in the relative gene expression. These results suggest that the short‐term exposure of mature bovine COCs to SIN‐1 does not induce stress tolerance and has no beneficial effect on bovine in vitro embryo production.
- The application of apoptotic inhibitor in apoptotic pathways of MII stage
porcine oocytes after vitrification
- Authors: Y Niu; J Dai, C Wu, Y Chen, S Zhang, D Zhang
Abstract: Apoptosis is one of the main drivers of the decline in developmental potential of porcine oocytes after vitrification. However, which apoptotic pathways are engaged after vitrification remains poorly understood. To distinguish among the possible apoptotic pathways induced by vitrification of MII stage porcine oocytes, this study detected activity and expression levels of several key proteins and genes in both the death receptor and mitochondrial pathways using in situ fluorescence staining and real‐time PCR (RT‐PCR) following the addition of specific inhibitors of either the death receptor or the mitochondrial apoptotic pathway (Z‐IETD‐FMK or Z‐LEHD‐FMK, respectively) into the incubation solution. Survival and parthenogenetic developmental ability were also examined. The results showed the following: (i) compared with the vitrified group, the activities of pan‐caspase, caspase 3, caspase 8 and caspase 9 as well as the early apoptotic rate were significantly lower in the Z‐IETD‐FMK and Z‐LEHD‐FMK groups (p
- Effect of climate region and stocking density on ostrich (Struthio
camelus) productive performances
- Authors: M Bouyeh; A Seidavi, H Mohammadi, A Sahoo, V Laudadio, V Tufarelli
Abstract: The effects of three climates (hot and dry, mild and humid and Alpine) and three flock densities (300 m2) on ostrich reproductive and productive traits were studied. Data were compared with the benchmark target sets by the World Ostrich Association (Ostrich benchmark Performance Targets. Version 2, May, 2008) for reproductive qualifications of ostrich. No significant difference was observed on egg production, weight, fertility, hatchability and day‐old chicks weight among the three climate conditions; however, the Alpine climate had a lower performance trend. Mild and humid climates had a significant effect of age at sexual maturity for both males and females as well as on the duration of egg production season. Stocking density did not show significant difference on egg production, hatchability, age of male and female at sexual maturity and on duration of egg production season, while an area >300 m2 showed a reduction in egg weight and day‐old chick weight. Further, an area
- Expression and localization of angiopoietin family in corpus luteum during
different stages of oestrous cycle and modulatory role of angiopoietins on
steroidogenesis, angiogenesis and survivability of cultured buffalo luteal
- Authors: SR Mishra; MS Parmar, VP Yadav, R Reshma, J Bharati, MK Bharti, A Paul, VS Chouhan, G Taru Sharma, G Singh, M Sarkar
Abstract: The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P4) secretion and mRNA expression of phosphotidylinositide‐3kinase‐protein kinase B (PI3K‐AKT), phosphoinositide‐dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real‐time RT‐PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p
- Effects of duration of electric pulse on in vitro development of cloned
cat embryos with human artificial chromosome vector
- Authors: LTK Do; M Wittayarat, T Terazono, Y Sato, M Taniguchi, F Tanihara, T Takemoto, Y Kazuki, K Kazuki, M Oshimura, T Otoi
Abstract: The current applications for cat cloning include production of models for the study of human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a human artificial chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC‐transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.
- Expression of retinoic acid‐metabolizing enzymes, ALDH1A1, ALDH1A2,
ALDH1A3, CYP26A1, CYP26B1 and CYP26C1 in canine testis during post‐natal
- Authors: VR Kasimanickam
Abstract: Mammalian spermatogenesis involves highly regulated temporal and spatial dynamics, carefully controlled by several signalling processes. Retinoic acid (RA) signalling could have a critical role in spermatogenesis by promoting spermatogonia differentiation, adhesion of germ cells to Sertoli cells, and release of mature spermatids. An optimal testicular RA concentration is maintained by retinaldehyde dehydrogenases (ALDHs), which oxidize RA precursors to produce RA, whereas the CYP26 class of enzymes catabolizes (oxidize) RA into inactive metabolites. The objective was to elucidate gene expression of these RA‐metabolizing enzymes (ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, CYP26B1 and CYP26C1) and their protein presence in testes of young, peripubertal and adult dogs. Genes encoding RA‐synthesizing isozymes ALDH1A1, ALDH1A2 and ALDH1A3 and RA‐catabolizing isomers CYP26A1, CYP26B1 and CYP26C1 were expressed in testis at varying levels during testicular development from birth to adulthood in dogs. Based on detailed analyses of mRNA expression patterns, ALDH1A2 was regarded as a primary RA‐synthesizing enzyme and CYP26B1 as a critical RA‐hydrolysing enzyme; presumably, these genes have vital roles in maintaining RA homeostasis, which is imperative to spermatogenesis and other testicular functions in post‐natal canine testis.
- Multicystic degeneration of the Cowper's gland in a Large White boar
- Authors: A Grahofer; H Nathues, C Gurtner
Abstract: The present report describes a case of multicystic degeneration of the Cowper's gland in a 1.3‐year‐old purebred Large White intact boar with reduced fertility in Switzerland. Based on the case history, a general physical examination, an andrological investigation and a transrectal ultrasonography combined with a subsequent thorough pathological examination including histology a multicystic degeneration of the Cowper's gland were diagnosed. The case indicates that cystic degeneration of the bulbourethral gland should be contemplated in the differential diagnoses of andrological disorders even though it has not been described in pigs so far. While selecting breeding boars, a morphological check of the bulbourethral gland can be performed, since degeneration of the gland would potentially have an impact on future fertility.
- Does serum anti‐Müllerian hormone levels always discriminate presence
of the ovaries in adult bitches? Comparison of two ELISA kits
- Authors: I Pir Yagci; M Pekcan, IM Polat, H Kalender, HC Macun
Abstract: Anti‐Müllerian hormone (AMH) is produced in the ovary, and thus, it is an excellent marker of follicle pool in females. Current interest is the clinical use of this parameter as a biomarker to assess presence or absence of an intact ovary and to diagnose ovarian remnant syndrome (ORS) following incomplete ovariohysterectomy (OHE) in bitches. The aim of this study was to evaluate serum AMH concentrations in bitches (n = 34) before and after OHE using two different commercial ELISA kits, one of which is based on detecting human AMH and the other is based on detecting human AMH and the other specified for canine AMH. Furthermore, serum AMH levels were also measured in six ORS cases to compare the diagnostic utility of the two different ELISA kits. Serum AMH concentrations measured using the human and canine kit prior to and after OHE were 0.32 ± 0.24, 0.006 ± 0.22 ng/ml (p
- Twin reduction by PGF2α intraluteal instillation in dairy cows
- Abstract: The objective of this study was to determine whether induced luteolysis of one of the two corpora lutea in twin pregnancies would provoke spontaneous twin reduction. In Experiment 1, 12 post‐partum cows with two corpora lutea in the same ovary were assigned to (three cows per group): Group I, Group II, Group III or Group IV receiving into one of the corpora lutea puncture with no treatment, 0.5 mg dinoprost, 1.5 mg dinoprost and 2.5 mg dinoprost, respectively. One of the two corpora lutea showed clear signs of luteolysis on Day 2 and was practically non‐detectable on Day 7 after treatment in the three cows of the Group IV. In Experiment 2, 11 cows carrying live twins with two corpora lutea on Day 28 of gestation, eight bilateral and three unilateral, received 2.5 mg dinoprost into one of the corpora lutea. Corpus luteum reduction and embryo reduction after treatment were registered in 10 and 9 cows, respectively. In bilateral twin pregnancies, four cows suffering embryo reduction remained pregnant. In unilateral twin pregnancies, membrane detachment resulted in the death of both cotwins. In conclusion, although observations were based on few animals, there seems to be a mechanism that operates locally to transfer ovarian progesterone to the uterus, and also a quantitative relationship between the amount of progesterone secreted and support of conceptuses, resulting in death of one twin embryonic vesicle when one corpus luteum regresses.
- Hormonal changes and follicular activity after treatment with intravaginal
progesterone‐releasing devices in llamas
- Authors: MV Cavilla; CP Bianchi, F Aguilera, M Hermida, MA Aba
Abstract: Plasma progesterone (P4) concentrations and follicular activity after administration of different P4 doses were evaluated in 33 adult female llamas treated with intravaginal devices. In Study 1, a group of llamas (n = 10) was treated with an intravaginal device (IVD) containing 160 (n = 5) or 780 mg of P4 (n = 5). Based on the results from the first study, in Study 2, females with follicles at different stages of development were treated with the IVD containing 780 mg of P4 (n = 21) or remain untreated (control; n = 12) to evaluate the effect of P4 on follicular activity. In Study 1, the IVD containing 160 mg of P4 induced follicular turnover in 60% of females while the remaining 40% of llamas developed persistent follicles. Thus, this device controlled follicular activity in llamas, although it promotes the persistence of follicles present at start of treatment. Conversely, in both studies, the IVD containing 780 mg of P4 suppressed follicular development and hasten the emergence of a new follicular wave in all females regardless of the follicular phase at insertion. Additionally, in Study 2, this device effectively concentrated the appearance of follicles with ovulatory diameter at a definite time after treatment in comparison with control animals. In conclusion, treatment with an IVD containing 780 mg of P4 would be considered for the control of follicular activity in llamas as it ensures the presence of a young follicle with ovulatory diameter by day 6 after the end of treatment in all females.
- Selection of red deer spermatozoa with different cryoresistance using
- Abstract: The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post‐thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post‐thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll®, Puresperm® and Bovipure™, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post‐thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.
- GnRH and prostaglandin‐based synchronization protocols as alternatives
to progestogen‐based treatments in sheep
- Abstract: The study investigated, for cycling sheep, synchronizing protocols simultaneously to the standard “P” protocol using progestogens priming with intravaginal devices and gonadotropin. In November 2014, 90 adult Menz ewes were assigned to either the “P” protocol, “PGF” treatment where oestrus and ovulation were synchronized using two injections of prostaglandin 11 days apart or a “GnRH” treatment where the ewes had their oestrus and ovulation synchronized with GnRH (day 0)–prostaglandin (day 6)–GnRH (day 9) sequence. The ewes were naturally mated at the induced oestrus and the following 36 days. Plasma progesterone revealed that 92% of the ewes were ovulating before synchronization and all, except one, ovulated in response to the applied treatments. All “P” ewes exhibited oestrus during the 96‐hr period after the end of the treatments in comparison with only 79.3% and 73.3% for “PGF” and “GnRH” ewes, respectively (p
- Identification of candidate miRNAs and expression profile of yak oocytes
before and after in vitro maturation by high‐throughput sequencing
- Authors: XR Xiong; DL Lan, J Li, XD Zi, MY Li
Abstract: Small RNA represents several unique non‐coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II‐arrested stage (M II) and then sequenced using small RNA high‐throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR‐342 has the largest fold change (9.25‐fold). Six highly expressed miRNAs (let‐7i, miR‐10b, miR‐10c, miR‐143, miR‐146b and miR‐148) were validated by real‐time quantitative PCR (RT‐qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy‐five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division.
- Effects of donor cells’ sex on nuclear transfer efficiency and telomere
lengths of cloned goats
- Abstract: The aim of this study was to investigate the effects of donor cells’ sex on nuclear transfer efficiency and telomere length of cloned goats from adult skin fibroblast cells. The telomere length of somatic cell cloned goats and their offspring was determined by measuring their mean terminal restriction fragment (TRF) length. The result showed that (i) reconstructed embryos with fibroblast cells from males Boer goats obtained significantly higher kids rate and rate of live kids than those of female embryos and (ii) the telomere lengths of four female cloned goats were shorter compared to their donor cells, but five male cloned goats had the same telomere length with their donor cells, mainly due to great variation existed among them. The offspring from female cloned goats had the same telomere length with their age‐matched counterparts. In conclusion, the donor cells’ sex had significant effects on nuclear transfer efficiency and telomere lengths of cloned goats.
- Development of the cardiorespiratory system in dogs from days 16 to 46 of
- Abstract: Dogs have been studied for several reasons, such as the genetic improvement, their use as experimental models, in zoonotic research, cell therapy and as a model for human diseases. However, many features relating to the embryonic development of dogs remain unknown because of the absence of embryological studies. Considering the importance of the cardiorespiratory system in the development of embryos, the aim of this study was to investigate the development of the main cardiorespiratory organs of dog embryos and foetuses with estimated gestational ages from 16 to 46 days using macro‐ and microscopic descriptions. On day 16 of development, the neural tube and crest were formed, the anterior and posterior neuropore closure had begun and the somites had developed. Between days 22 and 27 of gestation, the lung buds and the initial formation of the primary bronchi and heart chambers were observed. The heart chambers exhibited the endo‐, myo‐ and epicardial layers but did not have obvious differences in thickness among each other. Between days 41 and 46 of gestation, the nasal conchae and septa and trachea were formed, which exhibited characteristic epithelia. The lung formation and lobation were complete. The heart and major vessels exhibited mature histological architecture when their anatomical development was complete. The results of this study contribute to a more accurate definition of the embryonic and foetal developmental stages in dogs.
- Effects of concanavalin A on the progesterone production by bovine
steroidogenic luteal cells in vitro
- Abstract: The aim of this study was to evaluate the effects of concanavalin A (CONA) on the progesterone (P4) production by bovine steroidogenic luteal cells (LCs) in vitro. Luteal cells were collected during the mid‐luteal stage (at 10–12 days following ovulation) and processed in the laboratory. Luteal cells were grown for 7 days in a humid atmosphere with 5% CO2, with or without 10% foetal bovine serum, and were subjected to the following treatments: control: no treatment; CONA (10 μg/ml); LH (100 μg/ml); CONA + LH; LH (100 μg/ml) + prostaglandin F2α (PGF2α) (10 ng/ml); CONA + LH + PGF2α. Samples of the culture media were collected on days 1 (D1) and 7 (D7) for P4 quantification. The cells were counted on D7 of culture. Differences between treatments were considered statistically significant at p
- Metabolic blood profile of beef heifers during oestrous and
- Authors: EM Crane; JC Munro, SL Bourgon, M Diel de Amorim, R Ventura, AH Fredeen, YR Montanholi
Abstract: Haematological metabolic profiles in heifers could contribute to the development of proxies for oestrous detection and provide clues to further characterize biological changes during oestrus. One hundred and seven beef heifers were observed for oestrous behaviour twice daily for 124 days. Feed intake and productive performance (body weight and composition) traits were measured, and feed efficiency was determined using residual feed intake (kg DM/day). Blood plasma samples were collected when signs of oestrus were observed and every 30 ± 2 days. Heifers were considered in oestrus (n = 71) when plasma progesterone concentrations were
- Relationship between endometritis and oxidative stress in the follicular
fluid and luteal function in the buffalo
- Authors: BK Behera; CG Sharma, SK Singh, H Kumar, RK Chaudhari, AS Mahla, GK Das, N Krishnaswamy
Abstract: In this study, alteration in the follicular fluid composition and luteal function was investigated in the buffalo with endometritis. Genitalia were classified into cytological and purulent endometritis on the basis of polymorphonuclear cell cut off while non‐endometritis served as control (n = 10/group). In the follicular phase, the number of surface follicles was counted, diameter of the largest follicle was measured and the follicular fluid was assayed for total protein, cholesterol, malondialdehyde (MDA), total antioxidant capacity (TAC), oestradiol (E2) and progesterone (P4). The P4 content of corpus luteum during mid‐luteal phase was estimated by radioimmunoassay. Ovaries from the follicular phase of oestrous cycle showed no significant difference in the total number of surface follicles, size of the largest follicle and volume of follicular fluid in the buffaloes with and without endometritis (p > .05). However, the antral fluid of the largest follicle from the genitalia of buffalo with cytological and purulent endometritis showed a significant decrease in the concentration of total protein, cholesterol, TAC and E2 and a significant increase in the concentration of MDA and P4 (p
- Effect of growth differentiation factor‐9 (GDF‐9) on the progression
of buffalo follicles in vitrified–warmed ovarian tissues
- Abstract: To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor‐9 (GDF‐9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF‐9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen–thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF‐9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF‐9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF‐9 was detected in mural granulosa cells and theca cells of pre‐antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF‐9. In corpora lutea, GDF‐9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF‐9 and cultured control that indicated the GDF‐9 treatment has no effect on the primordial to primary follicle transition. GDF‐9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre‐antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF‐9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF‐9. In conclusion, treatment with GDF‐9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).
- The benefits of cooling boar semen in long‐term extenders prior to
cryopreservation on sperm quality characteristics
- Abstract: This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep® Plus (AHP), Androstar® Plus (ASP), Safecell® Plus and TRIXcell® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1−/PI−) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.
- Genomewide analysis of bull sperm quality and fertility traits
- Authors: R Puglisi; G Gaspa, D Balduzzi, A Severgnini, R Vanni, NPP Macciotta, A Galli
Abstract: Because the priority of AI industry is to identify subfertile bulls, a predictive model that allowed for the prediction of 91% bulls of low fertility was implemented based on seminological (motility) parameters and DNA status assessed both as DNA fragmentation index (DFI) and by TUNEL assay using sperm of 105 Holstein–Friesian bulls (four batches per bull) selected based on in vivo estimated relative conception rates (ERCR). Thereafter, sperm quality and male fertility traits of bulls were explored by GWAS using a high‐density (777K) Illumina chip. After data editing, 85 bulls and 591,988 SNPs were retained for GWAS. Of 12 SNPs with false discovery rate
- Elevating glucose and insulin secretion by carbohydrate formulation diets
- Authors: TY Chen; D Lines, C Dickson, C Go, RN Kirkwood, P Langendijk
Abstract: Primiparous (P1) sows commonly lose excessive body reserves to meet energy requirements for maintenance and milk production during lactation, and consequently, post‐weaning reproductive performance may be compromised. The present studies determined whether ad libitum feeding a glucogenic carbohydrate diet (CHO) during late lactation could stimulate insulin and glucose secretion (experiment 1) and improve subsequent litter size (experiment 2). For experiment 1, 15 P1 sows, and for experiment 2, 99 P1 sows (198.5 ± 2.7 kg) were allocated randomly according to suckled litter size (≥10 piglets), either to a CHO diet (14.3 MJ DE/kg, 19.8% crude protein) or a standard lactation diet (control; 14.2 DE MJ/kg, 19.5% crude protein) at 8 days before weaning. The CHO diet aimed to provide glucogenic content (extruded wheat, dextrose and sugar) as energy sources instead of fat sources without changing total dietary energy. Pre‐prandial plasma glucose and insulin concentrations were not influenced by treatments. However, post‐prandial plasma glucose and insulin concentrations and their peaks were both higher (p .05). Second litter size was not influenced by diet (p > .05), but the weaning‐to‐mating interval was shorter in CHO sows (p
- Mechanistic target of rapamycin is activated in bovine granulosa cells
after LH surge but is not essential for ovulation
- Abstract: The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR‐1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH‐induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.
- Dynamics of sperm DNA fragmentation in raw boar semen and fertility
- Authors: C Batista; E Lier, H Petrocelli
Abstract: The aims were to evaluate sperm DNA fragmentation (SDF) in boars through the dispersion of their chromatin in raw semen samples, quantifying the extent of SDF, and to assess dynamic aspects of sperm DNA damage after incubation to obtain the rate of sperm DNA fragmentation (rSDF) under thermal conditions similar to the uterus (37°C) over a period of up to 24 hr and to correlate the reproductive outcome of the sows with the SDF of the boars at ejaculation. The study was performed on a pig‐breeding farm in southern Uruguay. Sixty‐one ejaculates from five of the most frequently used hybrid boars were evaluated. Semen was collected weekly from each of the boars, using the gloved‐hand technique and discarding the jelly‐like fraction of the ejaculate. Fresh semen was kept in a water bath at 37°C and protected from light, and was thereafter processed with Sperm‐Sus‐Halomax® to evaluate SDF. The smears for time 0 (T0) were made on farm, and thereafter smears were made at the laboratory at 4 hr of obtaining the semen (T4), then every 2 hr (T6, T8, T10, T12) and a final fixation at 24 hr (T24). Differences in SDF were observed among exposure times for all boars (p
- The first case of genetically confirmed monozygotic twinning in the dog
- Abstract: Monozygotic twinning has not previously been genetically confirmed in the dog. This case report describes the finding of two viable male monozygotic foetuses within one placental site during caesarean section. Their umbilical cords attached to a single placenta. Genetic profiling using a total of 38 microsatellite markers, as well as amelogenin and SRY for sex determination, revealed identical DNA profiles, whether derived from blood or tissue (buccal swabs) samples. To the best of our knowledge, this is the first report of monozygotic twinning in the dog confirmed using DNA profiling.
- Influence of different anaesthetic protocols over the sperm quality on the
fresh, chilled (4°C) and frozen‐thawed epididymal sperm samples in
- Authors: M Batista; J Vilar, I Rosario, E Terradas
Abstract: This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine–dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50–100 × 106 spermatozoa ml−1, 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p
- Oestrous sheep serum balances ROS levels to supply in vitro capacitation
of ram spermatozoa
- Abstract: Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z‐VAD‐FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer‐assisted semen analysis (CASA) and viability (propidium iodide), apoptotic‐like membrane changes (YO‐PRO‐1), acrosomal status (PNA‐FITC), intracellular calcium (FLUO‐3), membrane fluidity (M540) and ROS production (CM‐H2DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p
- Laminin‐111 Inhibits Bovine Fertilization but Improves Embryonic
Development in vitro, and Receptor Integrin‐β1 is Involved in
- Abstract: This study detected the distribution of laminin during embryonic formation by immunofluorescence. To determine the possible function of laminin on developmental ability of in vitro fertilized embryos, the presumptive zygotes were divided and transferred to CR1aa medium supplemented with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml) of laminin. To explore the association with sperm–oocyte fusion, oocytes and/or sperm were pre‐incubated with laminin or anti‐β1 antibody before insemination. Laminin was absent in mature oocytes and could be detected first at the 8‐cell stage and then displayed an increasing tendency. Adding 10 μg/ml laminin to the culture medium improved embryonic development including cleavage rate, blastocyst rate, total cell numbers in the blastocyst and cell numbers in the inner cell mass. Laminin inhibited sperm–oocyte fusion when incubated with oocytes and/or sperm before in vitro fertilization, and only integrin‐β1 of sperm was involved in sperm–oocyte binding. Inhibition may be caused by blocking β1, but why laminin inhibits fertilization is still unknown. The results suggest that laminin plays an important role during embryonic formation and has a negative function in sperm–oocyte fusion, but improves embryonic development. However, only integrin‐β1 is involved in sperm–oocyte binding.
- Distribution of inflammation and association between active and chronic
alterations within the endometrium of dairy cows
- Authors: O Bogado Pascottini; M Hostens, P Dini, J Vandepitte, R Ducatelle, G Opsomer
Abstract: Objectives of this study were twofold: (i) to assess the association between polymorphonuclear (PMN) counts and chronic alterations within the bovine endometrium and (ii) to determine the distribution of inflammation throughout the endometrium of clinically healthy dairy cows. Holstein‐Friesian cows (n = 32) from a single dairy farm were selected for this experiment. Before slaughtering, a complete reproductive examination was performed to discard any type of clinical disease. After slaughtering, reproductive tracts were collected, and the endometrium was sampled at 8 pre‐defined locations. At each location, endometrial biopsies (EBs) and cytology (CY) samples were harvested. Histopathology samples were stained with haematoxylin–eosin (EB‐HE) and naphthol‐AS‐D‐chloroacetate‐esterase (EB‐naphthol), while CY samples were stained with Wright–Giemsa. In the EB‐HE samples, parameters assessed were epithelium height, mononuclear cells infiltration, lymphocytic aggregates, periglandular fibrosis, angiosclerosis and haemorrhage. In EB‐naphthol and CY slides, PMNs counts were evaluated. Binomial logistic regression was used to assess the association between the number of PMNs present in both the EB‐naphthol and CY samples and alterations identified in the EB‐HE samples and to analyse the distribution of the histopathological alterations (EB‐HE). A Poisson mixed‐effect model was used to analyse the distribution of PMNs within the endometrium. A significant positive association was found between the PMN counts and the mononuclear cells infiltration. The presence of erythrocytes was associated with higher odds to detect PMNs in the stratum compactum. Significantly, higher infiltration of PMNs and mononuclear cells were detected in the uterine body and the right horn region. Concluding, CY is a technique that allows the evaluation of PMN counts and therefore only evaluates active inflammation. A complete assessment of endometrial health can only be obtained using EB. To optimize the sensitivity to diagnose endometrial inflammation in cows, adjacencies of the corpus uteri should be considered as the preferred region to harvest samples.
- A review on prolificacy genes in sheep
- Abstract: Ovulation rate and litter size are important reproduction traits in sheep and are of high economic value. Reproduction traits typically have low to medium heritabilities and do not exhibit a noticeable response to phenotypic selection. Therefore, inclusion of genetic information of the genes associated with reproductive ability could efficiently enhance the selection response. The most important major genes affecting prolificacy and their genetic diversities in different sheep breeds were reviewed. Different causative mutations with major effects on reproductive traits including ovulation rate and litter size have been found in various sheep breeds around the world. A general overview of the studies on main prolificacy genes showed that some alleles may express different phenotypic effects in different breeds, and thus, further studies on epistatic effects are necessary for more understanding of genetic control of reproductivity in sheep. Regarding the polygenic control of fertility traits, application of new high‐throughput technologies to find new variants is essential for future studies. Moreover, genomewide association studies and genomic best linear unbiased predictions of breeding values are likely to be effective tools for genetic improvement of sheep reproductive performance traits.
- Expression pattern of matrix metalloproteinases changes during
folliculogenesis in the cat ovary
- Authors: M Fujihara; K Yamamizu, DE Wildt, N Songsasen
Abstract: Matrix metalloproteinase (MMP) has been implicated as having roles in ovarian folliculogenesis. Here, we determined the expression pattern of six MMPs (MMP1, MMP2, MMP3, MMP7, MMP9 and MMP13) and their endogenous tissue inhibitor, TIMP1, during cat follicle growth. Different developmental stage follicles were mechanically isolated and gene expression analysed by real‐time qPCR while MMP1, 2, 9 and 13 localization was determined by immunohistochemistry. With the exception of MMP13, the amount of MMP mRNA was lowest in primordial follicles and increased thereafter. Peak levels were detected in early antral follicles for MMP1 (72.2‐fold increase above primordial follicle amount), MMP2 (10‐fold), MMP3 (57‐fold) and MMP9 (2.8‐fold). MMP7 transcripts increased 2‐fold by the primary follicle stage and then plateaued. MMP13 mRNA peaked in primary follicles (2.5‐fold) and was lower in more advanced counterparts. TIMP1 sharply increased (6‐fold) in secondary follicles and gradually declined in the later stages. MMP1 and MMP9 expression were expressed in the granulosa cells of all follicle stages. MMP2 was immunoreactive in early and antral follicles, especially at granulosa cells adjacent to the antral cavity. By contrast, the MMP13 was weakly detected in primary follicles onward. In summary, there are distinctive and consistent changes in MMPs and TIMP1 expression during follicle development, suggesting that these enzymes play one or more roles in cat folliculogenesis. In particular, high mRNA and protein expression levels of MMP1 and MMP2, especially at the antral stage, indicate that these enzymes likely are involved in antrum formation and expansion.
- Exogenous paraoxonase‐1 during oocyte maturation improves bovine
embryo development in vitro
- Abstract: Paraoxonase‐1 (PON1) is an enzyme found in serum and follicular fluid that protects cell membrane and circulating lipids against oxidative damage. The aims of this study were to measure the direct effects of recombinant PON1 (rPON1) on bovine oocyte maturation at the molecular level (gene expression) and to measure the carry‐over effects of PON1 on pre‐implantation embryo development in vitro. COCs were submitted to IVM with the addition of 0.0, 0.02, 0.04 and 0.08 mg ml−1 of rPON1, corresponding to an average PON1 arylesterase enzyme activity of 2.2 ± 0.4, 15.5 ± 1.5, 30.2 ± 3.0 and 57.9 ± 5.0 U ml−1, respectively. The results indicated that addition of rPON1 during IVM improved embryo development in a dose‐dependent manner as D7 embryo development was 22.2%, 29.4%, 32.2% and 37.0% for the treatment groups, respectively (p = 0.02). In conclusion, addition of PON1 enzyme during IVM exerted dose‐related positive effects on embryo development rates to blastocysts.
- Monitoring the reproductive physiology of six‐banded armadillos
(Euphractus sexcinctus, Linnaeus, 1758) through different techniques
- Authors: LB Campos; GCX Peixoto, GL Lima, TS Castelo, AM Silva, CIA Freitas, AR Silva
Abstract: The aim of this study was to monitor the oestrous cycle using vaginal cytology, ultrasound and measurement of hormone levels associated with the modification of external genitalia in female Euphractus sexcinctus. Five adult female six‐banded armadillos were used for the study. Every three days, we chemically restrained the animals with a combined dose of ketamine and xylazine for 90 days. On each occasion, we conducted vaginal cytology and monitored the alterations in the vulval appearance. In addition, we obtained blood samples for serum estradiol and progesterone analysis and evaluated the ovaries by ultrasonography (8 MHz). As results, at least two entire cycles were monitored per female as based on external oestrous signs. We determined that six‐banded armadillos' oestrous cycle lasts 23.5 ± 3.12 days, comprising 8.8 ± 1.4 days for oestrogen phase, in which we verified vaginal bloody discharge, vulvar oedema, presence of mucus and ease of introduction of the swab. During oestrus, females presented an oestrogen peak of 240.66 ± 12.69 pg ml−1, on average, with a positive visualization of ovary follicles by ultrasound. The progesterone phase lasts 15.62 ± 2.1 days, characterized by the absence of bloody secretion and difficulty in introducing the swab; there was verification of a progesterone plateau of 10.83 ± 1.86 ng ml−1, on average, with identification of corpora lutea in 60% of the ovaries. This is apparently the first description of the six‐banded armadillos' oestrous cycle, which proves the efficiency of a multiparametric analysis to monitor it.
- Transcriptome analyses of inner cell mass and trophectoderm cells isolated
by magnetic‐activated cell sorting from bovine blastocysts using single
- Abstract: Research on bovine embryonic stem cells (bESCs) has been hampered because bESCs are cultured in conditions that are based on information obtained from culturing mouse and human inner cell mass (ICM) cells. The aim of this study was to compare gene expression in ICM and trophectoderm (TE) cell lineages of bovine embryos and to discuss the findings relative to information available for mice and humans. We separated a high‐purity (>90%) ICM and TE from bovine blastocysts by magnetic‐activated cell sorting and analysed their transcriptomes by single cell RNA‐seq. Differentially expressed genes (DEGs) were assessed using Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases. Finally, qRT‐PCR was performed to validate the RNA‐seq results. From 207 DEGs identified (adjusted p ≤ .05; fold change ≥2), 159 and 48 had greater expression in the ICM and TE cells respectively. We validated 27 genes using qRT‐PCR and found their expression patterns were mostly similar to those of RNA‐seq, including 12 novel ICM‐dominant (HNF4A, CCL24, FGFR4, IFITM3, PTCHD2, GJB5, FN1, KLK7, PRDM14, GRP, FGF19 and GCM1) and two novel TE‐dominant (SLC10A1 and WNT4) genes. Bioinformatics analysis showed that these DEGs are involved in many important pathways, such as MAPK and cancer cell pathways, and these pathways have been shown to play essential roles in mouse and human ESCs in the self‐renewal and pluripotent maintenance. As a conclusion, there were sufficient differences to allow us to conclude that the control of pluripotency in bovine ICM cells is species‐specific.
- Feto‐maternal heart rate ratio in pregnant bitches: effect of
gestational age and maternal size
- Authors: S Alonge; M Mauri, M Faustini, GC Luvoni
Abstract: Few information is available on parameters that can be used to objectively assess the foetal health during canine pregnancy. To identify a reliable parameter for the evaluation of foetal well‐being, the effect of pre‐gestational maternal bodyweight and gestational age on foetal heart rate (FHR) and on feto‐maternal heart rate ratio (FHR/MHR) was investigated. Seventeen client‐owned pregnant bitches of different pre‐gestational maternal bodyweight were examined by serial echo colour Doppler. Only data from 11 uncomplicated pregnancies were included in the statistical analysis. The relationship between FHR, and FHR/MHR, and independent variables was analysed by polynomial regression (p ≤ .05). The FHR and the FHR/MHR significantly fitted a multiple quadratic regression for all independent variables. They both increased from 35 to 20 days before parturition and then a decreasing pattern followed. Higher values of both parameters were observed in bitches of lowest and highest bodyweight. Patterns of FHR and FHR/MHR were similar, but the ratio better describes the effect of the independent variables on the data. Thus, the highest significance of FHR/MHR compared to FHR alone encourages the application of this ratio to evaluate foetal well‐being. The equation derived by the regression analysis of FHR/MHR could be applied in clinical practice to obtain its expected values in healthy pregnancies.
- Reliable collection of Caspian brown trout (Salmo trutta caspius) sperm
using a catheter
- Authors: MS Aramli; K Golshahi, A Banan, E Sotoudeh
Abstract: The traditional stripping procedure for collecting fish semen is associated with the risk of urine contamination, which may significantly affect semen quality and quantity. The use of a catheter as an alternative method for semen collection may overcome this problem. Therefore, this study compared Caspian brown trout (Salmo trutta caspius) semen parameters (i.e. sperm density, seminal plasma osmolality, motility parameters of spermatozoa analysed using computer‐assisted sperm analysis and fertility) between the traditional stripping method and the use of a catheter. All parameter values of the semen collected with a catheter were significantly higher (p
- Effects of low‐density lipoproteins as additive on quality parameters
and oxidative stress following cryopreservation of mithun (Bos frontalis)
- Authors: P Perumal; SK Srivastava, SK Ghosh, KK Baruah, S Bag, JS Rajoria, K Kumar, C Rajkhowa, M Pande, N Srivastava
Abstract: Artificial breeding of mithun poses several challenges including lack of standard protocol for cryopreservation of spermatozoa. This is further complicated by harmful effects of hen's egg yolk (EY) as additive in extender. Purified low‐density lipoproteins (LDL) extracted from EY have been shown as beneficial over EY extender for long‐term semen storage in several species. This investigation explored use of LDL versus EY on semen quality and oxidative stress following freezing–thawing of spermatozoa. A total of 25 of 50 ejaculates based on biophysical parameters were selected for the experiment. After diluting with the Tris‐citrate‐glycerol (TCG) extender, each sample was split into three equal aliquots: Group I, control, EY; Group II and Group III contained 8% and 10% purified LDL, respectively. Frozen–thawed samples were evaluated for motility parameters (progressive, and in the bovine cervical mucus penetration test [BCMPT]), viability, sperm and nuclear abnormality, acrosome integrity, and enzymatic (leakage of intracellular contents) and biochemical (oxidative stress) profiles and in vitro fertility (IVF) assay. Study revealed a significant (p
- The use of gelatine in long‐term storage (up to 48 hr) at 5°C
preserves the pre‐freezing and post‐thawing quality of brown bear
- Abstract: Sedimentation of spermatozoa occurs during long‐term liquid storage and this may produce deleterious changes. Our aim was to apply gelatine supplementation during long‐term pre‐freezing storage of bear sperm, applying final dilution and 6% glycerol at room temperature and cool in straws. We tested four models of sperm storage using a 1:1 dilution in TTF‐ULE‐Bear extender (TesT‐fructose‐egg yolk‐glycerol 6%): (i) second 1:1 dilution at room temperature (RT), cooling at 5°C in a tube and final dilution (100 × 106 sperm ml−1) (Standard); (ii) final dilution at RT and cooling in a tube (FD‐Tube); (iii) final dilution at RT and cooling in 0.25 ml plastic straw (FD‐Straw); and (iv) final dilution at RT in extender supplemented with 1.5% gelatine (Gelatine) and cooling in a 0.25 ml plastic straw. A Standard sample was stored at 5°C for 1 hr (Control); the rest of the samples (Standard, FD‐Tube, FD‐Straw, Gelatine) were stored for 24 or 48 hrs before freezing (100 × 106 sperm ml−1, glycerol 6%). The quality of the samples was assessed for motility by CASA, and viability (SYBR‐14/propidium iodide‐PI‐; VIAB), acrosomal status (PNA‐FITC/PI; iACR) and apoptotic status (YO‐PRO‐1/PI; YOPRO‐) by flow cytometry. At pre‐freezing, after 48 hr, Gelatine showed significantly higher viability (for VIAB and YOPRO‐) and progressiveness (PM, LIN and STR). At 48 hr, Gelatine showed similar YOPRO‐, iACR, LIN, STR and ALH respect to Control. At both 24 and 48 h post‐thawing, Gelatine sample had similar scores for YOPRO‐, iACR, LIN, STR, WOB and VIAB (only 24 hr) when compared with Control, and lower for TM, PM, rapidPM, VAP and ALH. No differences were found among others experimental groups with respect to Control. In conclusion, gelatine could be a suitable alternative to preserve the viability and progressive motility of brown bear ejaculates during long‐term pre‐freezing storage at 5°C.
- Effect of estradiol cypionate and GnRH treatment on plasma
estradiol‐17β concentrations, synchronization of ovulation and on
pregnancy rates in suckled beef cows treated with FTAI‐based protocols
- Abstract: Two experiments were conducted to evaluate the effect of different ovulation inducers on E‐17β plasma concentrations, synchronized ovulations and pregnancy rates. In Experiment 1, cows received a progesterone intravaginal device (PID) with 1 g of progesterone (P4) plus 2 mg of estradiol benzoate (EB) (day 0). At PID removal (day 8), cows received 0.150 mg of D‐cloprostenol and were randomly assigned to four treatment groups (n = 10/treatment): Group ECP: 1 mg of estradiol cypionate at PID removal, Group EB: 1 mg of EB 24 hr after PID removal, Group GnRH: 10 μg of GnRH 48 hr after PID removal, Group ECP‐GnRH: 1 mg of ECP at PID removal plus 10 μg of GnRH 48 hr later. Ultrasonographic examinations were performed to detect the dominant follicle and ovulation. GnRH‐treated cows ovulated later (p
- Long‐term Effects of Pyrethrin and Cyfluthrin, a Type II Synthetic
Pyrethroid, Insecticide Applications on Bull Reproductive Parameters
- Authors: JL Stewart; CF Shipley, FA Ireland, VL Jarrell, CL Timlin, DW Shike, TL Felix
Abstract: The objectives of this study were to determine effects of cyfluthrin and pyrethrin spray products, used in combination with cyfluthrin topical and ear tag applications, on bull reproductive parameters over 18 weeks. Angus or Angus x Simmental bulls were randomly assigned to one of three treatment groups: (i) no exposure to pyrethrins/cyfluthrin (CONT; n = 10), (ii) cyfluthrin ear tag and topical applications (ET; n = 10), or (iii) cyfluthrin ear tag, topical, premise spray and pyrethrin fog spray applications (ET+S; n = 8). Bull body weight was measured every 3 week, and body condition score and scrotal circumference were recorded on weeks 0, 9 and 18. Semen and serum were collected every 3 weeks for sperm evaluation and testosterone measurement, respectively. There was a treatment × week interaction (p
- Inhibition of FGF Signalling Pathway Augments the Expression of
Pluripotency and Trophoblast Lineage Marker Genes in Porcine
- Authors: LY Li; MM Li, SF Yang, J Zhang, Z Li, H Zhang, L Zhu, X Zhu, V Verma, Q Liu, D Shi, B Huang
Abstract: The consistent failure to isolate bona fide pluripotent cell lines from livestock indicates that the underlying mechanisms of early lineage specification are poorly defined. Unlike other species, the contrivances of segregation have been comprehensively studied in the mouse. In mouse, FGF/MAPK signalling pathway dictates the segregation of hypoblast (primitive endoderm). However, it is not evident whether this mechanism is also conserved in livestock. Here, in this study, we examined the roles of FGF/MAP kinase signalling pathways in porcine parthenogenetic embryos during the early development. Porcine parthenogenetic embryos were cultured in the medium addition with FGFR inhibitor BGJ398 (10 μm) or DEMOS. Pluripotency‐ and lineage‐related gene expressions in the early porcine embryos were determined. Compared to control, total cell numbers on day 7 were significantly higher (55 ± 5.96 vs 47 ± 1.97, p 0.05). Nonetheless, BGJ398 treatment significantly augmented the expression of pluripotency and trophoblast marker genes (SOX2, OCT4, KLF4 and CDX2), but did not significantly change the expression of NANOG and hypoblast marker gene (GATA4). Furthermore, the addition of FGF signalling agonist (FGF2) during the embryo development significantly decreased the expression of pluripotency and trophoblast marker genes (SOX2, NANOG, KLF4 and CDX2), but no significant effect on the expression of OCT4 and GATA4 was observed. Here, we exhibit that inhibition of FGF signalling could improve the quality of the porcine embryo and escalate the chance to capture pluripotency. Besides, it also promotes the trophoblast development of porcine parthenogenetic embryo. In addition, the data suggested that FGF signalling pathway is dispensable for the segregation of hypoblast and epiblast lineages in porcine embryo during the early development.
- Impact of Food Restriction on the Expression of the Adiponectin System and
Genes in the Hypothalamic–Pituitary–Ovarian Axis of Pre‐Pubertal
- Authors: R Wang; M Kuang, H Nie, W Bai, L Sun, F Wang, D Mao, Z Wang
Abstract: Adiponectin, a cytokine secreted typically by adipocytes, has been implicated as a molecular switch between female reproduction and energy balance. The present study was undertaken to investigate the expression of adiponectin system and patterns of genes in the hypothalamic–pituitary–ovary (HPO) axis of food‐restricted pre‐pubertal ewes. Eighteen 2‐month‐old female ewes were assigned to 3 groups after a pre‐feeding ad libitum for 10 days (six in each group): the control group (C), the low‐food‐restricted group (LR) and the high‐food‐restricted group (HR), which were fed with 100%, 70% and 50% of ad libitum food intake, respectively. The hypothalamus, pituitary, ovary and serum were collected after food restriction for 2 months. Results by ELISA showed that food restriction increased serum adiponectin concentrations. Quantitative real‐time PCR showed that the gene transcriptions for adiponectin receptor 1 (AdipoR1) and 2 (AdipoR2) were enhanced in the hypothalamic–pituitary–ovarian (HPO) axis, while KISS‐1/GPR‐54 and gonadotropin‐releasing hormone (GnRH) in the hypothalamus and luteinizing hormone β‐subunit (LHβ) and follicle‐stimulating hormone β‐subunit (FSHβ) in the pituitary were reduced after food restriction. Immunohistochemistry results demonstrated that AdipoR1 localized in the oocytes of follicles in the ovary. These results suggest that the alterations in the expression of adiponectin and its receptors in response to food restriction might negatively influence the HPO axis.
- Membrane Stress During Thawing Elicits Redistribution of Aquaporin 7 But
Not of Aquaporin 9 in Boar Spermatozoa
- Abstract: Freezing of boar spermatozoa includes the cryoprotectant glycerol, but renders low cryosurvival, owing to major changes in osmolarity during freezing/thawing. We hypothesize that aquaporins (AQPs) 7 and 9 adapt their membrane domain location to these osmotic challenges, thus maintaining sperm homeostasis. Western blotting (WB) and immunocytochemistry (ICC) at light and electron microscope levels with several commercial primary antibodies and protocols explored AQP location on cauda epididymal and ejaculated spermatozoa (from different fractions of the ejaculate), unprocessed, extended, chilled and frozen‐thawed. Although differences in WB and ICC labelling were seen among antibodies, AQP‐7 was conspicuously located in the entire tail and cytoplasmic droplet in caudal spermatozoa, being restricted to the mid‐piece and principal piece domains in ejaculated spermatozoa. AQP‐9 was mainly localized in the sperm head in both caudal and ejaculated spermatozoa. While unaffected by chilling (+5°C), freezing and thawing of ejaculated spermatozoa clearly relocated the head labelling of AQP‐7, but not that of AQP‐9. In vitro mimicking of cell membrane expansion during quick thawing maintained the localization of AQP‐9 but relocated AQP‐7 towards the acrosome. AQP‐7, but not AQP‐9, appears as a relevant marker for non‐empirical studies of sperm handling.
- Issue Information
- Pages: 629 - 630