Animal Reproduction, Volume: 17, Issue: 1, Published: 2020
  • Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts Original Article

    Nascimento Júnior, Agnelo Douglas do; Marques, Juliana Carla Cavalcanti; Cezar, Allan Rodolf Ribeiro; Batista, André Mariano; Kastelic, John Patrick; Câmara, Diogo Ribeiro

    Abstract in English:

    Abstract Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.
  • Chlorogenic acid improves the quality of boar semen processed in Percoll Original Article

    Rabelo, Stenia Severo; Resende, Carla Oliveira; Pontelo, Thais Preisser; Chaves, Bruna Resende; Pereira, Bárbara Azevedo; Silva, William Eduardo da; Peixoto, Juliano Vogas; Pereira, Luciano José; Zangeronimo, Márcio Gilberto

    Abstract in English:

    Abstract This study aimed to evaluate if the addition of chlorogenic acid (ChA) to semen extenders improves the quality of cooled boar semen processed in Percoll. The experimental design was randomized blocks (ejaculates) in a 2×3 factorial (with or without Percoll, and three antioxidant systems: a negative control, without supplementation, a positive control – vitamin E, and ChA), totaling six treatments and 12 repetitions. ChA and vitamin E (VE) were added at 4.5 mg/ml and 400 μg/ml in extender, respectively. At 0, 48 and 72h of storage at 15ºC, 80 ml insemination doses each containing 2.0 billion sperm cells were submitted to centrifugation in Percoll. The use of Percoll impaired (P<0.01) all motility patterns but decreased (P<0.01) the number of abnormal cells at 0, 48 and 72h of storage. Both VE and ChA improved (P<0.05) the total motility after Percoll processing, but only in semen stored for 48h. The same effect was not observed (P>0.05) in semen stored for 72h. ChA improved (P<0.05) the total motility of the semen stored for 72h, but this effect was not observed (P>0.05) when the semen was processed in Percoll. The antioxidants had no effect (P>0.05) on the viability and integrity of the acrosome, but ChA reduced (P<0.05) the number of abnormal cells at 0h, while VE increased the number of abnormal cells in semen stored for 72h, independent of the use of Percoll. There was no effect (P>0.05) of antioxidants or Percoll on the concentration of malondialdehyde in seminal plasma. The use of Percoll had no effect (P>0.05) on the cholesterol efflux, but ChA increased (P<0.05) this parameter at 0h and reduced (P<0.05) in the semen stored for 72h not processed with Percoll. In conclusion, the addition of ChA to semen extenders improved the quality of boar semen processed or not in Percoll.
  • Subclinical endometritis and postpartum ovarian resumption in respect to TNF-α, IL-8 and CRP in Egyptian buffaloes Original Article

    Elsayed, Doaa H.; El-Azzazi, Fakhri E.; Mahmoud, Yasmina K.; Dessouki, Sherif M.; Ahmed, Eman A.

    Abstract in English:

    Abstract The study was carried out to find the relation between subclinical endometritis (SCE) and postpartum (pp.) ovarian resumption as well as to evaluate serum and endometrial TNF-α, IL-8 and serum CRP in buffaloes with and without SCE. Thirty-nine pluriparous buffaloes at the 3rd (W3), 5th (W5) and 7th (W7) week pp. were involved in this experiment. The parity of the buffaloes ranged from 4 to 8 with an average 5.8±0.2. Subclinical endometritis was diagnosed by the percentage of polymorphonuclear cells (PMNs) in uterine cytology obtained from endometrial cytobrush at W5 and W7. The cut-off point of PMNs% in buffaloes for SCE was ≥ 6% at W5 or ≥ 4% at W7. According to PMNs%, buffaloes were divided into SCE group (n=27) and non-SCE group (n=12). Ovarian cyclicity was monitored by rectal palpation, ultrasonography and progesterone assay at W3, W5 and W7. Serum and endometrial TNF-α, IL-8 and serum CRP were estimated at W5 and W7. Buffaloes with SCE (55.6%) showed delayed ovarian activity as compared to non-SCE (16.7%) animals (P=0.036). Significant increase in serum cytokines and CRP levels were detected at W5 (P ˂0.05) and W7 (P <0.01) in SCE buffaloes as compared to non-SCE. Endometrial levels of cytokines were significantly (P ˂0.05) elevated in SCE buffaloes. Serum and endometrial cytokines showed significant positive correlation. Furthermore, levels of TNF-α, IL-8 and CRP exhibited significant positive correlation with PMNs%. In conclusion, SCE delayed postpartum ovarian cyclicity in buffaloes. Moreover, TNF-α, IL-8 and CRP assessments could be efficient tools in prediction of SCE in buffaloes.
  • Effect of supplementation of Aloe vera extracts in cold storage media and cryopreservation of domestic cat epididymal spermatozoa Original Article

    Barbosa, Brenna de Sousa; Santos, Fernanda Araujo dos; Macêdo, Luãn Barbalho do; Izzo, Roberta Gonçalves; Fernandes, Denilsa Pires; Praxedes, Érika Almeida; Silva, Alexandre Rodrigues; Bezerra, Marcelo Barbosa

    Abstract in English:

    Abstract This study evaluated the effect of the extract of Aloe vera at concentrations of 10% and 20% on the cryopreservation of sperm from the epididymis of domestic cats. Epididymal spermatozoa were recovered using the flotation technique and used in the treatments: control (TRIS-egg yolk at 20%), T10% (TRIS plus 10% of A. vera extract), and T20% (TRIS plus 20% of A. vera extract). The spermatozoa were subjected to 4ºC for 60 minutes, followed by 20 minutes in nitrogen vapors, and stored in a cryogenic cylinder. The samples were thawed at 37°C for 30 seconds. The sperm motility decreased (P<0.05) after thawing in the three treatments. Only the spermatozoa in the control treatment maintained post-thawing vigor. The viability of spermatozoa decreased in the treatments with A. vera (P<0.05). According to the hypoosmotic test, all treatments maintained the sperm membrane functionality (P>0.05) during freezing; however, after thawing, it decreased (P<0.05) in the T10% and T20% treatments. The morphology and chromatin condensation of spermatozoa did not differ, regardless of the treatments and time of evaluation (P>0.05). The effect of the crude A. vera extract was not satisfactory on the cryopreservation of epididymal spermatozoa of domestic cats after thawing; although the motility of spermatozoa was similar to that found with the use of egg yolk, and it presented maintenance of the chromatin integrity. However, it is necessary to understand the action of the substances present in A. vera with the feline spermatozoa, well as the standardization and adjustment of physicochemical characteristics aiming at the future application of the vegetal extract.
  • Relationship between Body Mass Index (BMI) and testicular and hormonal parameters of sexually active male greater cane rats (Thryonomys swinderianus) Original Article

    Adebayo, Adenrele Olalekan; Akinloye, Adebayo Kuyoom; Oke, Bankole Olusiji; Taiwo, Victor Olusegun

    Abstract in English:

    Abstract The current upsurge in intensive farming practices of greater cane rat has not only lead to higher growth rate but is accompanied by increased fat deposition especially in the males. This study attempts to characterize one of the most commonly used fat estimation parameter, the body mass index (BMI) as well as evaluates its relationship with testicular and hormonal parameters in seventy-two sexually active male cane rats over a period of one year. Six animals, kidded and raised in a farm, with known ages were used each month. The experimental protocols entail body measurements of weight, height and length; histology; orchidometry; and hormonal immunoassay of testosterone, estradiol, progesterone, LH and FSH using their various kits. The mean values of the body mass (BMI) and Lee (LI) indices of male greater cane rats were 1.18±0.20g/cm2 and 0.30±0.02g/cm respectively with the testicular histology indicating normal spermatogenesis. BMI/LI, both of which followed the same pattern, neither correlate with testicular parameters nor with serum testosterone, progesterone, LH and FSH concentrations but had low correlations with serum estradiol concentration (r2 = 0. 2; p = 0.0023). So, these relationships may provide clue on obesity and its effect on reproductive performance and strengthened the possibility of the characterized BMI/LI as obesity marker for breeding selection in male cane rat.
  • Lectin spatial immunolocalization during in vitro capacitation in Tursiops truncatus spermatozoa Original Article

    Robles-Gómez, Laura; Fuentes-Albero, María del Carmen; Huerta-Retamal, Natalia; Sáez-Espinosa, Paula; García-Párraga, Daniel; Romero, Alejandro; Gómez-Torres, María José

    Abstract in English:

    Abstract Spermatozoa interactions with the female reproductive tract and oocyte are regulated by surface molecules such as glycocalyx. The capacitation process comprises molecular and structural modifications which increase zona pellucida binding affinity. Lectins allowed us to describe glycocalyx changes during maturation, capacitation and acrosome reaction. This study had as its aim to identify lectin binding patterns using four lectins with different carbohydrate affinity in bottlenose dolphin (Tursiops truncatus) spermatozoa both before and after in vitro capacitation. Two semen samples from the same dolphin obtained on consecutive days were used, with four different lectin binding patterns becoming visible in both samples before and after capacitation. A highly stained equatorial segment with prolongations at the edges appeared as the most frequent pattern with Wheat germ agglutinin (WGA) in uncapacitated spermatozoa. However, it was homogeneously distributed over the acrosomal region after capacitation. Instead, the use of Peanut agglutinin (PNA) resulted in most spermatozoa showing high labelling in the acrosomal periphery region before capacitation and a homogeneous staining in the acrosomal region within the population of capacitated spermatozoa. Nevertheless, the most representative patterns with Concavalin A (ConA) and Aleuria aurantia agglutinin (AAA) lectins did not change before and after capacitation, labelling the acrosomal region periphery. These findings could contribute to the understanding of the reproductive biology of cetaceans and the improvement of sperm selection techniques.
  • Testicular development induced by GnRH-IS in budgerigar (Melopsittacus undulatus) Original Article

    Herrera-Barragán, José Antonio; Carcoba-Pérez, Samantha Anahí; Pérez-Rivero, Juan José; Ávalos-Rodríguez, Alejandro; Vargas-Ibarra, Ana Karen; Gual-Sill, Fernando; López-Díaz, Osvaldo

    Abstract in English:

    Abstract Nowadays, the third part of parrots in the world is endangered or vulnerable; an alternative for their preservation is assisted reproduction in captivity through hormonal manipulation. In birds, GnRH is the main hormone which controls reproductive physiology, it is known there are three types: GnRH-I, GnRH-II and GnRH-III, involved in the release or inhibition of luteinizing hormone and follicle stimulant hormone to control gonadal and gametic development. The objective of this study was, to evaluate the effect of administrating synthetic GnRH-I in the testicular development of Melopsittacus undulatus. Twenty-eight adult budgerigars were randomly divided in two groups: control (n=14) and treated (n=14) with a unique dose of synthetic GnRH-I. Testicular development was assessed through ultrasonography and density was evaluated with pixels. Germinal diameter and thickness of germinal epithelium were determined with histology; there were identified and countified different cellular strains in seminiferous tubules therefore spermatobioscopy. Results. Ecographic density was: control group: 76 ± 7 pixels, treated group 41 ± 3 pixels. Thickness of germinal epitellium, 51.5 ± 2.9µm and 73.1 ± 3.1µm, for control group and treated group respectively. Sperm concentration in the treated group was 300% superior than in control group. It is concluded that the administration of synthetic GnRH-I, is a viable alternative to be used as part of the assisted reproductive techniques to induce reproduction.
  • Effect of temperature – humidity index on the onset of post- partum ovarian activity and reproductive behavior in Bos indicus cows Original Article

    Díaz, Ramiro Fernando; Galina, Carlos Salvador; Aranda, Emilio Manuel; Aceves, Lorenzo Armando; Sánchez, Jaime Gallegos; Pablos, Jose Luis

    Abstract in English:

    Abstract The effect of climatic factors on ovarian activity and reproductive behavior (RB) was evaluated in 46 Bos indicus cows kept under grazing conditions. Temperature-humidity index (THI) was used as an indicator of stress and divided in alert, damage and emergency levels. Fat thickness (FAT) was taken during the last trimester of gestation (LTG) to approximately 90d postpartum (PP). At 30d PP animals received a progesterone (P4)-releasing device (CIDR) which was withdrawn 9d later. Ovarian activity was assessed by blood progesterone on days 21, 24, 27, 30, 49, 51, and 54 PP. Animals were divided into three groups, higher, and moderate RB and non-behavior. Sixty percent presented a THI >74 increasing dramatically from June to September up to >78. During LTG, animals lost 27% of their body reserves contrasting to PP where an increase of 2.6% (P=0.002) was observed. The percentages of cyclic and non-cyclic animals were 57 and 43%, respectively (P> 0.05). Seventy-two percent displayed RB and 28% were non-behavior (P<0.05). A negative correlation (r = -0.307; P = 0.038) between THI and RB, and a positive correlation (r = 0.427; P = 0.003) between the onset of ovarian activity and RB were observed. Differences in THI during the LTG (P<0.01) were observed between cyclic and non-cyclic animals. Non-behavior cows in the LTG had a higher THI (P <0.05). High levels of THI have a negative effect on the resumption of ovarian activity and RB in Bos indicus especially if high THI occurs during the last trimester of gestation.
  • Wild Leporinus friderici induced spawning with different dose of mGnRHa and metoclopramide or carp pituitary extract Original Article

    Souza, Thiago Gonçalves de; Kuradomi, Rafael Yutaka; Rodrigues, Suely Marlene; Batlouni, Sergio Ricardo

    Abstract in English:

    Abstract Breeding technology is of utmost importance for reproduction of wild fish in captivity for the reintroduction and selective breeding programs purposes. The main challenge is that when applied to wild undomesticated specimens, conventional protocols often cause breeders and/or embryo mortality and spawning failure. In this study, we evaluated the reproductive performance of wild Leporinus friderici, a great importance fish for subsistence fishing in South American rivers, applying conventional and lower-dose hormonal therapies by means of two consecutive experiments. In the first, a conventional (0.5 and 5.5 mg/kg) and a lower carp pituitary extract (CPE) dose (0.5 and 1.0 mg/kg) were applied. In the second, a conventional mammalian GnRH analogue associated with metoclopramide (mGnRHa + MET) (40 µg mGnRHa + 20 mg MET/kg) and a lower dose (4 μg mGnRHa + 2 mg MET/kg and 8 µg + 4 mg of mGnRHa + MET/kg) were applied. Ovulation was observed in all treatments, however, only lower CPE protocol provided viable embryos. High levels of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17β estradiol (E2) detected in conventional, but not in lower CPE dose, at ovulation, might be associated to the mortality of the embryos. The use of lower CPE dose applied here was the best way to obtain L. friderici viable embryos. These results directly contribute to the knowledge about poorly explored effects of reproductive management and hormonal therapies in wild-type breeders, showing that the use of reduced doses may be an alternative to reproductive success.
  • The gynecological examination in Pantaneiro mares Original Article

    Milan, Bruno; Sampaio, Breno Fernandes Barreto; Silva, Eliane Vianna da Costa e; Leal, Cassia Rejane Brito; Navarro, Bruna; Gomes, Danilo Carloto

    Abstract in English:

    Abstract Gynecological examination is essential to assess the reproductive tract of mares and can provide important information about the uterine environment. It includes physical, vaginal, and rectal examination, ultrasound, cytology, culture, and endometrial biopsy. The present study aimed to perform gynecological examination and fertility to assess the fertility prognosis of Pantaneiro mares that have not been reproductively active and to determine their reproductive ability. Eight mares underwent ultrasound and gynecological examination and artificial insemination. Ultrasound revealed changes only in one mare. Histopathological findings were mild, such as periglandular and perivascular inflammatory cell infiltrates, fibrotic areas, glandular dilation, glandular island formation, and edema due to the phase of the estrous cycle. One animal was classified in category I and the others in category IIA. Cytological changes were found in only one mare. Endometrial culture from five mares resulted in isolation of Pseudomonas sp., Bacillus sp., Escherichia coli and Candida sp. Only four mares resulted pregnant through artificial insemination, using the same stallion with fresh semen, which has been proving fertility. Thus, mares with better uterine conditions will not always become pregnant and those with mild changes will not always be barren.
  • Change in plasma sirtuin 1 level by injection into uterus of resveratrol in Korean cattle Original Article

    Kim, Dae Hyun; Yi, Jun Koo; Ha, Jae Jung; Oh, Dong Yep; Kim, Do Yeoung; Lee, Yoon seok

    Abstract in English:

    Abstract Bovine embryonic development is closely associated with mitochondrial biogenesis, which is regulated by the sirtuin 1 (SIRT1). Resveratrol, which is a type of natural phenol produced by several plants and used as a dietary supplement, is the activator of SIRT1. Although it has been reported that resveratrol increased SIRT1 level in in vitro bovine blastocysts, there are no in vivo reports on the change in the plasma SIRT1 level in cows. Therefore, we investigated the change in the level of plasma SIRT1 by injecting different concentrations of resveratrol into the uterus of Korean cattle heifer. The level of plasma SIRT1 in the 1.0 μM resveratrol-injected group was the highest among all groups (P < 0.05). Although the level of plasma SIRT1 increased on days 7, 9, and 14 in the resveratrol-injected group, the level of plasma SIRT1 in the control group decreased. When 1.0 µM resveratrol was directly injected into the uterus of cows during artificial insemination, a pregnancy rate was 21.0% higher than that in the control group. In conclusion, our results identified that the level of plasma SIRT1 was increased by direct injection of resveratrol and improved conception rate by injection into uterus of cow during artificial insemination.
  • Effects of the cryopreservation process on dog sperm integrity Original Article

    Sicherle, Carmen Cecilia; Souza, Fabiana Ferreira de; Freitas-Dell’Aqua, Camila de Paula; Mothé, Gabriele Barros; Padovani, Carlos Roberto; Papa, Frederico Ozanam; Lopes, Maria Denise

    Abstract in English:

    Abstract Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P <0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.
  • Current and alternative trends in antibacterial agents used in mammalian semen technology Review Article

    Santos, Caio Sérgio; Silva, Alexandre Rodrigues

    Abstract in English:

    Abstract The use of antibacterial substances as additives in extenders for ensuring the sanitary quality of the semen employed in reproductive biotechniques and preserving it from bacterial deterioration has been reported since the mid-twentieth century. However, the deleterious effects of these drugs on the sperm quality as well as their effectiveness in controlling bacterial growth in the preserved semen have been questioned. The aim of this review was to report the antimicrobials primarily used in the extenders added to the semen of mammals, and to present alternatives to their use. Among the various mammalian species, there is a large variation regarding the antimicrobial types added to semen extenders as cephalosporins (ceftiofur, cefdinir, eg) and quinolones (ofloxacin, ciprofloxacin), alone or in combination with large action spectra substances as penicillin-streptomycin and gentamicin-tylosin-lincomycin-spectinomycin. To combat problems related to bacterial resistance to these drugs, the emergence of alternatives is increasingly evident. Among these alternatives, use of physical methods as centrifugation and filtration, as well as the use of antimicrobial peptides and other substances from different origins have been highlighted for presenting antimicrobial potential.
  • Reproductive performance of sows inseminated with semen doses stored for up to seven days in long-term extender in a field condition Short Communication

    Lucca, Matheus Schardong; Ulguim, Rafael da Rosa; Bortolozzo, Fernando Pandolfo; Wentz, Ivo; Rocha, Janaina Colecha; Souza, Mayara Corrêa Dias de; Escobar, Roberto Vasconcelos; Calderam, Kérlin; Quadros Filho, Pedro Ivo de; Marcos, Raquel Ausejo

    Abstract in English:

    Abstract This study aimed to evaluate the reproductive performance of sows inseminated with semen doses preserved at 15-18 °C for up to seven days in long-term extender (Duragen®). Parity one (PO1) to PO7 sows were randomly assigned to the following groups: AI1-3 (n=190), insemination with semen doses stored between one and three days; and AI5-7 (n=124), insemination with semen doses stored between five and seven days. Sows were submitted to estrus detection twice a day. Post-cervical insemination according to weaning-to-estrus interval was performed. The farrowing rate (FR) did not differ between the groups (AI1-3=83.2%; AI5-7=82.2%; p>0.05) nor did the total number of piglets born (TPB; AI1-3=14.2±0.3; AI5-7=14.5±0.3; p>0.05). Considering the semen dose most likely responsible for fertilization according to its storage time (1, 2-3, 5, and 6-7 days), the FR (72.7%, 87.8%, 85.7%, and 79%, respectively) and TPB (14.4, 14.0, 14.9, and 13.5, respectively) were similar among the groups (p>0.05). In conclusion, the use of semen doses extended with long-term extender stored for up to seven days did not impair the reproductive performance of sows. Therefore, it’s using could optimize production efficiency and logistics of semen dose deliveries to sow farms.
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