RESUMEN

The aim of this study was to evaluate two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) in two concentrations (5 and 7 %) in vitro in donkey semen using a rapid freezing technique and the effect on pregnancy rates in mares. Twenty-four ejaculates from 8 jacks (n = 8; r = 3) were divided into 4 extenders: BotuSemen Gold with 5 % or 7 % MF and 5 % or 7 % DMF, all containing 11 % lactose, 20 % egg-yolk and 0.5 % Equex. Post-thaw evaluations included: sperm motility, membrane function and acrosome status. A linear mixed effect model was used to test the effect of different freezing media on semen parameters. No differences were observed between the 4 freezing media used, for any of the seminal parameters (P > 0.05). However, samples with 5 % DMF showed the highest percentages of sperm with acrosomes and functional membranes (DMF: 5 %: 53.67 ± 22.01; 7 %: 33.92 ± 23.4; MF: 5 %: 44.5 ± 20.46; 7 %: 38.75 ± 27.4) (Data: mean ± SD; P > 0.05). Hence, thirty mares were inseminated: 15 with 5 % DMF and 15 with 7 % DMF. The pregnancy rate was 46 % (7/15) and 0 % (0/15) using the extender with 5 % or 7 % DMF, respectively (P = 0.003). To conclude, the use of 5 % or 7 % of MF or DMF did not affect the in vitro parameters. Despite the lack of differences in vitro with the two DMF concentrations, in vivo results only showed pregnancies when using 5 % DMF. Thus, the results of this study demonstrate the importance of accompanying in vitro semen evaluations with studies that evaluate post-insemination pregnancy rates.

Asunto(s)

Criopreservación , Crioprotectores , Equidae , Preservación de Semen , Animales , Equidae/fisiología , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Crioprotectores/farmacología , Femenino , Masculino , Criopreservación/métodos , Criopreservación/veterinaria , Embarazo , Dimetilformamida/farmacología , Inseminación Artificial/veterinaria , Semen/efectos de los fármacos , Semen/química , Motilidad Espermática/efectos de los fármacos , Formamidas

RESUMEN

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 106 spermatozoa ml-1 and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra-rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6-carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.

Asunto(s)

Criopreservación/veterinaria , Preservación de Semen/instrumentación , Preservación de Semen/métodos , Preservación de Semen/veterinaria , Sus scrofa , Acrosoma/ultraestructura , Animales , Cruzamiento , Membrana Celular/fisiología , Supervivencia Celular , Cromatina/química , Cromatina/fisiología , Criopreservación/instrumentación , Criopreservación/métodos , Crioprotectores , Calor , Masculino , Análisis de Semen/veterinaria , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Motilidad Espermática , Espermatozoides/fisiología , Espermatozoides/ultraestructura , Sus scrofa/genética

RESUMEN

Owing to current problems in boar sperm cryopreservation, this study proposes to evaluate vitrification in spheres as an alternative cryopreservation procedure, comparing the use or not of permeable cryoprotectants and two warming methods. Extended (n = 3; r = 4) and raw (n = 5; r = 2) porcine spermatozoa were diluted in media, in the absence or presence of either 4% dimethylformamide or 4% glycerol, to a final concentration of 5 × 106  spermatozoa/ml and vitrified using the spheres method. Two warming procedures were evaluated: a rapid method (30 s at 37°C) and an ultrarapid method (7 s at 75°C, followed by 30 s at 37°C). Percentages of total motility (phase contrast), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), sperm viability (6-carboxyfluorescein diacetate and propidium iodide stain), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated in the samples before and after vitrification. Results, analysed using Friedman's test, suggest that rapid warming of raw porcine spermatozoa vitrified without permeable cryoprotectants may preserve DNA condensation and integrity better than the other processing methods studied in this work. Hence, porcine sperm vitrification using spheres could be used to produce embryos with ICSI to further validate this method.

Asunto(s)

Criopreservación/veterinaria , Preservación de Semen/veterinaria , Espermatozoides/efectos de los fármacos , Vitrificación , Animales , Supervivencia Celular/efectos de los fármacos , Criopreservación/métodos , Crioprotectores/farmacología , Dimetilformamida/farmacología , Masculino , Análisis de Semen , Preservación de Semen/métodos , Motilidad Espermática/efectos de los fármacos , Espermatozoides/citología , Porcinos

RESUMEN

The objective of this study was to compare the efficiency of different sperm selection methods applied to the same llama ejaculate. Four treatments were compared: two variants of the swim up technique (with and without seminal plasma), and two different colloids, Androcoll-E-Large and Percoll(®). Using electroejaculation, 21 semen samples were obtained from 7 llama males (n=7, r=3). The ejaculates were incubated in a solution of 0.1% collagenase, to decrease thread formation, and then split into 4 aliquots: one aliquot was layered over a column of Androcoll-E-Large (SLC) and the second over a column of Percoll (45%). The third aliquot was deposited in a tube with culture medium and was incubated at a 45° angle for 30min at 37°C (SU1). The last aliquot was centrifuged to separate the spermatozoa and seminal plasma. The sperm pellet obtained was resuspended, and transferred to a tube with culture medium which was incubated at an angle of 45° for 30min at 37°C (SU2). Both aliquots SLC and P showed higher proportions of progressive motility and plasma membrane functionality (p≤0.05) than raw semen. There were no significant differences (p>0.05) in sperm viability and in normal spermatozoa between raw semen and treatments. Nevertheless, only SLC did not have a significant increase of bent tails. In conclusion SLC centrifugation would be the method of choice for selecting llama spermatozoa.

Asunto(s)

Camélidos del Nuevo Mundo/fisiología , Separación Celular/métodos , Animales , Masculino , Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Motilidad Espermática/fisiología , Espermatozoides/fisiología

RESUMEN

Endometrial expression of oestrogen receptor-α (ERα), progesterone receptor (PR) and cyclooxigenase-2 (COX-2) was evaluated in non-pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non-pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post-induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post-mating and by progesterone profile on day 12 post-mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX-2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non-pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post-induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX-2 expression was lower in pregnant than in non-pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF(2α) secretion during early pregnancy by decreasing the endometrial expression of COX-2 in the luminal epithelium of pregnant llamas.

Asunto(s)

Camélidos del Nuevo Mundo , Ciclooxigenasa 2/metabolismo , Endometrio/metabolismo , Receptor alfa de Estrógeno/metabolismo , Preñez , Receptores de Progesterona/metabolismo , Animales , Biopsia , Buserelina/administración & dosificación , Femenino , Fármacos para la Fertilidad Femenina/administración & dosificación , Luteólisis/efectos de los fármacos , Embarazo

RESUMEN

The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose-egg yolk (L-EY), (2) Tris-citrate-fructose-egg yolk (T-F-EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5 h and remained at that temperature during 24 h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L-EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A-D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12 × 10(6) live spermatozoa, cooled to 5°C and kept for 24 h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24 h. These groups (A-C) were inseminated between 22 and 24 h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24 h and AI was carried out within 2 h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation.

Asunto(s)

Camélidos del Nuevo Mundo/fisiología , Frío , Inseminación Artificial/veterinaria , Semen/fisiología , Animales , Femenino , Fertilidad , Inseminación Artificial/métodos , Masculino , Ovulación , Embarazo , Preservación de Semen/veterinaria , Factores de Tiempo

RESUMEN

Llama production in Argentina has increased, as the international interest in breeding this type of animals has grown in the last years. Considering the great polymorphism that llama spermatozoa present at evaluation using light microscopy, the aim of this study was to objectively evaluate llama sperm head morphometry using digital morphometric analysis. Five ejaculates from each of eight males were obtained to evaluate morphometric parameters of 8000 sperm heads stained with Tinción 15(®). The following average results were obtained for each parameter: size parameters: area 20.09 µm(2), length 6.60 µm, width 4.14 µm, equivalent circle diameter 5.06 µm, curve length 5.79 µm and curve width 3.48 µm; boundary parameters: perimeter 18.54 µm and convex perimeter 17.34 µm; and shape parameters: roundness 1.28 and elongation 1.59. Morphometric parameters of sperm head were compared between ejaculates of the same male and between males. Significant differences between ejaculates of the same male were found for all parameters evaluated (P < 0.01). Significant differences between males were found for all morphometric parameters (P < 0.01) except for curve length, curve width and perimeter. The differences detected would indicate that there is not a single morphometric pattern for Lama glama sperm head, because parameter values cannot be standardised.

Asunto(s)

Camélidos del Nuevo Mundo , Cabeza del Espermatozoide/ultraestructura , Animales , Masculino

RESUMEN

The aim of the present study was two-fold. Experiment I: evaluate the effect of buserelin on llama's oocyte maturation after exogenous follicular activity suppression, followed by ovarian superstimulation with different doses of equine chorionic gonadotropin (eCG). Experiment II: compare the number of follicles aspirated and the number of cumulus-oocyte complexes (COCs) recovered according to different doses of eCG followed by buserelin. Experiment I consisted in a control group (without buserelin) and a treatment group (with buserelin), both subdivided according to eCG dose administered: A: 500 IU; B: 1000 IU; C: 1500 IU. The treatment group received a single i.v. dose of 8 microg of buserelin when two or more dominant follicles were found at ultrasound evaluation and 20 h later were subjected to surgery. In group A, 83% of the llamas did not respond to superstimulation. In groups B and C differences were observed between the control and the treatment groups for the degree of COCs maturation (p < 0.05). In experiment II animals were divided into two groups according to the eCG dose administered: 1000 and 1500 IU. Twenty hours before surgery females received a single i.v. dose of 8 microg of buserelin. Average number of follicles aspirated and COCs recovered was higher (p < 0.05) with the administration of 1500 IU of eCG. A larger number of expanded COCs were obtained from follicles > or =7 mm in diameter. We conclude that buserelin aids the recovery of a larger number of expanded COCs. Administration of 1500 IU of eCG produces a higher number of follicles for aspiration and number of COCs recovered.

Asunto(s)

Buserelina/administración & dosificación , Camélidos del Nuevo Mundo/fisiología , Gonadotropina Coriónica/administración & dosificación , Células del Cúmulo/fisiología , Fármacos para la Fertilidad Femenina/administración & dosificación , Oocitos/crecimiento & desarrollo , Animales , Células del Cúmulo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Caballos , Oocitos/efectos de los fármacos , Folículo Ovárico/diagnóstico por imagen , Inducción de la Ovulación/veterinaria , Succión/veterinaria , Recolección de Tejidos y Órganos/métodos , Recolección de Tejidos y Órganos/veterinaria , Ultrasonografía

RESUMEN

The aim of the present study was two-fold. First, to evaluate the effect of exogenous progesterone on ovarian follicular dynamics in order to assess its ability to synchronize ovarian activity in the vicuna. Secondly, to evaluate the ovarian response to the treatment with eCG through the observation of the structures developed in the ovaries. Follicular dynamics was monitored daily by transrectal ultrasonography in 12 adult, non-pregnant vicunas. Plasma progesterone and estradiol-17beta concentrations were measured in blood samples collected daily. In experiment 1, intravaginal devices containing 0.33g of progesterone were inserted into the vagina and kept in place for 5 days (treatment group, n = 8). After progesterone withdrawal, five animals were further monitored in order to evaluate the efficacy of the CIDR to synchronize the emergence of a dominant follicle. In experiment 2, four females received 750IU of eCG IM. Two were previously monitored ultrasonographically to confirm the absence of a dominant follicle at the beginning of the superstimulatory treatment (group A). The other two animals had a CIDR inserted into the vagina for 5 days and the superstimulatory treatment was applied 24h after device withdrawal (group B). Females from both groups were surgically explored 96 h after eCG injection; the ovaries were exposed and the number of newly formed structures produced by each ovary was counted. Peak progesterone concentrations (25.9 +/- 5.29 nmol l(-1), mean +/- S.E.M.) were attained on day 1 after device insertion, remained high until the day of device withdrawal (9.7 +/- 1.98 nmol l(-1)) and decreased to 5.5 +/- 1.13 nmol l(-1) the day after. There was no follicle development to the state of dominance after device insertion. Moreover, mean follicle diameter steadily decreased after insertion of the device until the minimum mean value (1.85 +/- 0.17 mm) was recorded on day 5 (P = 0.006). Similarly, plasma concentrations of estradiol-17beta remained below 35 pmol l(-1) during the period of progesterone treatment in all animals and the mean estradiol-17beta declined with the lowest value (22.1 +/- 2.19 pmol l(-1)) being recorded on day 4 after device insertion. After superstimulation of follicular development with eCG, the total number of follicles that developed was 33 in group A and 58 in group B and the mean number of newly developed ovarian structures per female was 22.75 +/- 4.26. In conclusion, progesterone released by the CIDR exerts a negative effect on ovarian follicular development and function suggesting intravaginal devices could be used to synchronize the beginning of follicular waves during a superstimulatory treatment. There was also a tendency for greater ovarian follicular development when the animals were previously treated with progesterone.

Asunto(s)

Camélidos del Nuevo Mundo/fisiología , Gonadotropinas Equinas/farmacología , Folículo Ovárico/efectos de los fármacos , Progesterona/farmacología , Animales , Camélidos del Nuevo Mundo/sangre , Estradiol/sangre , Femenino , Gonadotropinas Equinas/sangre , Folículo Ovárico/diagnóstico por imagen , Folículo Ovárico/fisiología , Progesterona/sangre , Ultrasonografía

RESUMEN

The objective of the present study was to characterize ovarian activity in non-mated vicunas, relating ovarian structures (evaluated by transrectal ultrasonography, daily for 30 days) to changes in plasma concentrations of estradiol-17beta and progesterone. Ovarian follicular activity occurred in waves, characterized by the follicle emergence, growth and regression. The mean duration of follicular waves was 7.2+/-0.5 days (mean+/-S.E.M.), with a range of 4-11 days. The follicular growth phase averaged 3.0+/-0.2 days, the static phase 1.4+/-0.1, the regression phase 2.9+/-0.3 days, and the inter-wave interval was 4.2+/-0.3 days. The mean growth rate during the growing phase was 1.8+/-0.1mm/day, while the duration of the interval from 6mm to maximum diameter was 1.4+/-0.1 days. The mean maximum diameter of the dominant follicle was 8.4+/-0.3mm (range: 6.2-11.2) and mean diameter of the largest subordinate follicle was 5.4+/-0.1mm. There was an inverse relationship between the size of the largest follicle and the total number of follicles (r=-0.21, P=0.002). Follicle activity alternated between ovaries in 77% of the waves, with 40% of dominant follicles present in the left ovary and 60% in the right ovary. Plasma estradiol-17beta concentrations also had a wave-like pattern, varying between 12.0 and 62.8 pmol/l. Plasma progesterone concentrations remained below 5.0 nmol/l and there was no ultrasonographic evidence of ovulation during the study.

Asunto(s)

Camélidos del Nuevo Mundo/fisiología , Estradiol/sangre , Folículo Ovárico/fisiología , Progesterona/sangre , Animales , Femenino , Ovario/diagnóstico por imagen , Ovario/fisiología , Factores de Tiempo , Ultrasonografía

RESUMEN

The hypo-osmotic test has been used successfully on equine semen and was considered to be a simple and accessible method which could be a useful addition to routine equine semen analysis. It was therefore of interest to determine whether the hypo-osmotic test is significantly correlated to proposed criteria of fertility. The stallions were divided into two groups on the basis of threshold levels of fertility. A significant difference (P<0.05) was found between the two groups for the following parameters: progressive motility, morphologically normal spermatozoa, percentage of swelling with the hypo-osmotic test, percentage of pregnant mares and number of services per pregnancy. The hypo-osmotic test provided a simple evaluation of membrane function and the results obtained show stallions with low swelling scores (<40%) to be of doubtful fertility. The hypo-osmotic test was not correlated with percentage of pregnant mares but showed a tendency to correlate with the number of services per pregnancy, therefore it could be an additional method for evaluating stallion fertility. Further studies are needed to confirm this observation.

Asunto(s)

Fertilidad , Caballos/fisiología , Espermatozoides/fisiología , Animales , Masculino , Ósmosis

RESUMEN

The occurrence of uterine and oviductal inflammation, and fertilisation rates, were measured on Day 3 post ovulation in inseminated mares that had either exhibited intrauterine lumenal fluid during a previous dioestrus (Experiment 1) or had acute endometritis induced by intrauterine infusion of 1% glycogen (Experiment 2). Endometritis was assessed by uterine cytology and histology whereas oviductal inflammation was measured histologically. Fertilisation rates were calculated from the percentage of cleaved ova recovered by retrograde flushing of the oviducts. Mares with or without pre-existing uterine fluid during dioestrus that were inseminated showed a higher incidence of endometritis than control mares without pre-existing uterine fluid that were not inseminated (n = 7 mares/group). However, inseminated mares with uterine fluid did not show a higher incidence of endometritis than inseminated mares without uterine fluid. Mares with or without pre-existing uterine fluid showed a higher incidence of endometritis than salpingitis and these 2 groups of mares showed equivalent rates of fertilisation and oviductal oocyte recovery. Mares inseminated with semen alone or semen following 1% glycogen treatment had a higher incidence of endometritis than control noninseminated mares (n = 17 mares/group) but mares that received semen plus 1% glycogen did not show a higher incidence of endometritis than mares that received semen alone. Both these groups of mares showed a higher incidence of endometritis than salpingitis and those that received semen plus 1% glycogen showed an equal recovery rate of recently ovulated ova but a lower fertilisation rate than the mares that received semen alone.

Asunto(s)

Endometritis/veterinaria , Fertilización In Vitro/veterinaria , Fertilización , Enfermedades de los Caballos/fisiopatología , Salpingitis/veterinaria , Útero/patología , Administración Intravaginal , Animales , Estudios de Cohortes , Endometritis/inducido químicamente , Endometritis/patología , Endometritis/fisiopatología , Femenino , Fertilización In Vitro/métodos , Glucógeno/administración & dosificación , Glucógeno/efectos adversos , Enfermedades de los Caballos/inducido químicamente , Enfermedades de los Caballos/patología , Caballos , Incidencia , Salpingitis/patología , Salpingitis/fisiopatología , Semen/fisiología

RESUMEN

The oxidative energy requirements of bovine spermatozoa capacitated with dilauroil-phosphatidylcholine liposomes (PC 12) and the effect of these liposomes on acrosome reaction necessary for in vitro fertilization were studied. Mitochondrial respiration was measured using 3 different substrates (pyruvate-lactate-glucose) and endogenous substrates. The samples were either treated with PC 12 or were left untreated and used as the control. A 2.8-fold increase in the consumption of oxygen was observed in the PC 12 treated spermatozoa in the presence of the 3 combined substrates (pyruvate-lactate-glucose). Respiration changes were not observed when the spermatozoa were capacitated with only 2 of the 3 substrates or with glucose alone. When endogenous substrates were used, the consumption of oxygen increased 1.7 times, and mitochondrial uncoupling was observed in the treated samples. The hypermotility characteristic of the capacitation process was not observed when glucose or endogenous substrates were used. When the percentage of intact acrosomes was determined using differential-interferential contrast (DIC) microscopy, it was found that in the presence of oxidative substrates there was a 26% decrease compared with that of the control sample. The proportion of reacted acrosomes was in the range of 41.3 to 49.6%, as measured by the chlortetracycline epifluorescence method in the presence of calcium ionophore A23187. Only 4% of the spermatozoa showed acrosome reaction with endogenous substrates. A higher percentage of fertilized oocytes were observed when the spermatozoa were capacitated in the presence of the 3 substrates (pyruvate-lactate-glucose), confirming that the success of in vitro fertilization depends on the energy conditions associated with the capacitation process. The results of these experiments indicate that the presence of oxidative energy is necessary to produce capacitation and the hyperactivation characteristic in frozen-thawed bovine spermatozoa treated with liposomes.