Lactoferrin increases sperm membrane functionality of frozen equine semen.

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 µg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 µM/µg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.

Evaluation of Fertilizing Ability of Frozen Equine Sperm Using a Bovine Zona Pellucida Binding Assay.

BACKGROUND: Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. OBJECTIVE: This work aimed at developing an assay to evaluate the fertilizing ability of frozen equine sperm using a bovine ZBA with the use of an in vitro capacitation and hyperactivation media with procaine and calcium ionophore A23187, respectively. MATERIALS AND METHODS: The sperm motility characteristics, intact and acrosome reacted sperm rates, and number of stallion sperm bound to the bovine ZP were calculated. RESULTS: The procaine group showed a hyperactivation motility pattern, although it improved ZP sperm binding similarly to the capacitation group. CONCLUSION: The capacitation medium improved the IVF capability of frozen equine sperm, allowing the highest possibility of sperm-oocyte interaction.

Detecting reproductive system abnormalities of broiler breeder roosters at different ages.

The objective of this study was to detect the reasons of rooster's fertility decrease at 50 weeks of age. Therefore, the reproductive system of broiler breeder roosters was laparoscopic, macroscopic and histopathology evaluated, and a comparison of the anatomical aspect with the sperm analysis and birds' age was realized. Cobb roosters (n = 59) were distributed into two groups (30 and 50 weeks). Evaluations were performed with laparoscopy, macroscopy and histopathology, and seminal quality, blood serum testosterone concentration and weight were also determined. The old roosters presented smaller testicle size, higher intensity epididymal lithiasis and lower testicle sperm production, compared to the young roosters. The use of the endoscope could easily distinguish a normal-sized testicle than an atrophic one. Four old roosters with severe testicular atrophy did not show spermatogenesis, although three still had sperm in the ejaculate. This would falsely indicate a wrong diagnosis of normal fertility before the testicular atrophy took place. In conclusion, in addition to the weight increase with age, the testicular atrophy and impairment of sperm production seemed to be the main reason to the decrease in the rooster's fertility at 50 weeks of age. Therefore, the use of the laparoscopy as a way to detect the roosters with testicular atrophy before 50 weeks of age and their removal from them flock could be useful as a diagnostic tool to prevent the birds' fertility loss.

Milk, caseinate and lactoferrin addition to equine semen cooling extenders.

Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 µg ml-1 , C4) C2 + Lf 500 µg ml-1 and C5) C2 + Lf 1000 µg ml-1 extenders, and kept at 5 °C for 24 h. Sperm motility characteristics and intact membrane rates were not different among the treatments (P > 0.05). As a result of the cooling process, the nitrite concentration increased significantly in the cooled semen (69.6 ± 78.9 µm per ×106 spermatozoa) compared with the fresh semen (8.6 ± 1.9 µm per ×106 spermatozoa). In contrast, the H2 O2 concentrations were lower in the 0.6% caseinate extender (265.9 ± 221.3 µm per ×106 spermatozoa) than in the milk extender (430.9 ± 199.8 µm per ×106 spermatozoa, P < 0.05), showing an antioxidative effect of the caseinate compared with the milk. However, in all groups, hydrogen peroxide concentrations were similar to the undiluted fresh semen (332.8 ± 151.3 µm per ×106 spermatozoa). Caseinate showed to be as efficient as milk to protect equine-cooled spermatozoon.

Comparison of conventional freezing and vitrification with dimethylformamide and ethylene glycol for cryopreservation of ovine embryos.

The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re-expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re-expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p < 0.05). The number of dead cells in vitrified embryos of Gr. 2 and Gr. 3 was higher (42.6 ± 26.2 and 63.2 ± 34.65, respectively) in comparison with Gr. 1 (conventional freezing, 10.1 ± 8.5, p < 0.05). Embryos vitrified with dimethylformamide included the same quality of apoptotic cells that Gr. 1 (conventional freezing) and fresh embryos. In conclusion, the dimethylformamide and ethylene glycol used as cryoprotectant to vitrify ovine embryos, in the concentrations and exposition time tested in this work, were not as efficient as the conventional freezing for cryopreservation of ovine embryos Thus, the conventional freezing with ethylene glycol was the most efficient method to cryopreserve ovine embryos in comparison with vitrification.

Caseinate protects stallion sperm during semen cooling and freezing.

Extenders with a defined composition containing only components with clearly protective effects on sperm during storage would be an advantage. The aims of the present work were to assess whether caseinate, improves cooled and frozen equine semen quality. Semen from six stallions were suspended with four different cooling extenders C1) Kenney extender; C2) 0.6 % caseinate; C3) 2.7 % caseinate ; and C4) C1 + 2.1 % caseinate, and frozen extenders: F1) INRA 82 extender; F2) 1.35 % caseinate; and F3) 2.7 % caseinate. Although there was no significant difference between the motility rate among the cooled (C1:45.0, C2:36.7, C3:38.3 and C4:48.3) and frozen extenders (F1:16.9, F2:21.1 and F3:18.6), significant higher values of sperm velocity variables were observed with the 1.35 % caseinate extender compared to the control (VSL: 40.8 x 18.9 and VAP: 46.8 x 25.0 µm/s), respectively. Caseinate seemed to be responsible for sperm protection during preservation and showed to be as efficient as milk.

Metabolic evaluation of cooled equine spermatozoa.

Microscopy has been used in the routine evaluation of sperm metabolism. Nevertheless, it has limited capacity to preview male fertility. As calorimetry may be used to evaluate directly the metabolic activity of a biological system, the aim of this study was to use microcalorimetry as an additive method for sperm metabolism evaluation of cooled equine semen. Two ejaculates of four stallions were collected and motility, viability (eosin 3%) and membrane functional integrity (hyposmotic swelling test) of spermatozoa were evaluated. Sperm samples were processed following different protocols and the metabolism of these samples was accessed by calorimetry. Centrifugation is part of some of these processing protocols and although this procedure has been deleterious for sperm viability and plasma membrane integrity, no decrease in sperm motility was observed. Microcalorimetry was capable of detecting the positive effect of re-suspending the sperm pellet with Kenney extender. Thus, the use of microcalorimetry offered additional information for equine sperm metabolism evaluation and was efficient in detecting important information from sperm cell metabolism.

Addition of ficoll and disaccharides to vitrification solutions improve in vitro viability of vitrified equine embryo.

The aim of the present study was to evaluate the in vitro viability of equine embryos vitrified in three different solutions. Day 6 and 6.5 embryos were measured and morphologically evaluated. Only grade 1 or 2 morulae and early blastocysts were vitrified. Eighteen embryos were distributed in Group 1: 40 percent ethylene glycol in PBS, Group: 2 and 3: 40 percent ethylene glycol, 18 percent Ficoll, 0.3M sucrose or 0.3M trehalose in PBS, respectively. The vitrified embryos were loaded individually into 0.25 ml straws, which were cooled and immersed in liquid nitrogen. After warming at 20 degree C for 20s, the embryos were expelled out into 0.5M sucrose in PBS and transferred to PBS solution. The embryonic diameter was measured again and morphology and viability were evaluated with Propidium iodide and Hoechst 33258 dyes. Embryos vitrified with sucrose (19.2 percent) and trehalose (26.7 percent ) showed the highest percentage of viable cells and morphological quality.

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen.

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10(6) sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST(+) (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI(-)/PSA(-) (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.

Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm.

Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen-thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 x 10(6)spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 degrees C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl-beta-cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl-beta-cyclodextrin/50 x 10(6) sperm; and (T4) T3 with 0.156 mg methyl-beta-cyclodextrin/50 x 10(6) sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen-thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3+/-5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5+/-11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3+/-7.1% and T4: 11.8+/-9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm.

Addition of recombinant human growth hormone to in vitro maturation medium of bovine oocytes.

The aim of this study was to increase the bovine embryonic development rate, adding recombinant human growth hormone (rhGH) to maturation medium of bovine oocytes. Oocytes were matured for 24 h in TCM 199 Earle's salts and five treatments were developed: T1, 0.01 IU/ml of recombinant human follicle stimulating hormone (rhFSH); T2, 0.01 IU/ml of rhFSH + 100 ng/ml of rhGH; T3, 0.01 IU/ml of rhFSH + 1000 ng/ml of rhGH; T4, 100 ng/ml of rhGH; and T5, 1000 ng/ml of rhGH at 39 degrees C and 5% of CO(2) in air and saturated humidity. In vitro fertilization from cumulus-oocyte complexes was conducted in TALP-Fert medium (18-22 h) and spermatozoa were selected by Percoll gradient. Zygotes were incubated in SOFaaci medium in 5% of CO(2) in air, 5% of O(2) at 39 degrees C and saturated humidity for 11 days. There was no statistical difference in cleavage rate and embryo production on day 7 and day 9 among treatments. However, the hatching rate increased significantly in the T4 and T5 treatments (11.0 and 12.8%, respectively), compared with the T1 treatment (4.6%) (p < 0.05). Therefore, the rhGH addition to the oocyte maturation medium showed beneficial effects on the hatching rate of in vitro-produced bovine embryos.

Assessing equine sperm-membrane integrity.

The swelling of cells in a hypo-osmotic medium has been described as an important criterion for assessing the functional integrity of the sperm plasma membrane. The resistance of equine spermatozoa to osmolarity changes was studied by extending 98 semen samples collected from nine stallions in media at five osmolarities (300, 200, 150, 100, and 50 mOsmol l(-1)). The response of the cells was measured by the spermatocrit technique and eosin staining. Spermatocrit determines the increase on spermatozoal volume under hypo-osmotic conditions, a sign of functional integrity of sperm plasma membrane, whereas the eosin staining evaluates the viability of spermatozoa. A significant positive correlation (P<0.01) was observed between spermatocrit values and percentage of eosin-unstained cells. Spermatocrit measurements and eosin staining proved to be useful methods to evaluate the integrity of sperm plasma membrane under hypo-osmotic conditions and could be used as an additional criterion to predict semen preservation ability.

Influência da infusäo transcervical de plasma seminal ou de estrógeno na concepçäo, no ciclo estral e na ovulaçäo de porcas / Influence of transcervical infusion of seminal plasma or oestrogen on conception, oestrus cycle and ovulation in sows

O objetivo deste trabalho foi o de avaliar o desempenho de 211 porcas da linhagem Camborough 22 Marca Registrada, de diferentes ordens de parto, submetidas à infusäo transcervical de plasma seminal ou de estrógeno no início do estro. Após o desmame, elas foram examinadas duas vezes ao dia para a detecçäo do estro, na presença de um macho sexualmente maduro, e da ovulaçäo, com auxílio da ultrasonografia transcutânea. No início do estro as fêmeas receberam aleatoriamente uma infusäo transcervical de plasma seminal, de estrógeno ou permaneceram como grupo-controle. Todas as fêmeas foram inseminadas no turno seguinte à detecçäo do estro, recebendo no máximo três inseminaçöes com intervalos de 8 a 16h. No grupo-controle as fêmeas de primeiro parto ovularam mais precocemente que as fêmeas com maior número de partos (P<0,05). Os tratamentos näo influenciaram a duraçäo do estro e o momento da ovulaçäo. O número total de leitöes nascidos näo diferiu entre os tratamentos