José Antonio Dell'Aqua

Use of cholesterol-loaded cyclodextrin: An alternative for bad cooler stallions

During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of the acrosome reaction (ACR) and on the fertility rate of cooled sperm. In experiment 1, each ejaculate was divided into four groups: Control and treated with 1 (CLC-1), 1.5 (CLC-1.5), or 2 mg (CLC-2) of CLC/120 × 10(6) sperm and cooled for 48 hours at 5 °C. In experiment 2, each ejaculate was divided into four groups: Control and CLC-1.5 cooled at 15 °C or 5 °C for 24 hours. For experiment 3, GC and BC stallions were used, and the ejaculates were divided into control and CLC-1.5 cooled at 5 °C for 48 hours. According to experiment, the sperm kinetics (SK) and plasma membrane integrity (PMI) were analyzed before and after 24 and 48 hours of cooling. In experiment 4, the ejaculates were divided into four groups: Control and CLC-1.5 maintained at room temperature or cooled at 5 °C for 24 hours. Each group was incubated with ionophore calcium at 37 °C for 3 hours. The incidence of ACR was analyzed before and after 1, 2, and 3 hours of incubation. For experiment 5, two cycles of 10 mares for a GC stallion and two cycles of 25 for a BC stallion were used. The inseminations were performed with control and CLC-1.5 groups cooled at 5 °C for 24 hours. According to results, all groups treated with CLC exhibited higher PMI compared with controls, and CLC-1.5 and CLC-2 exhibited the best SK results. The cooling temperature of 5 °C was superior to 15 °C when the sperm was treated with CLC. The GC and BC stallions benefited from the CLC-1.5 treatment, but the BCs were more evident, which presented greatly increased PMI and SK. There was a delay in capacitation of at least 3 hours for the fresh sperm and at least 1 hour for cooled sperm supplemented with CLC-1.5. After adding CLC-1.5, the fertility of BC stallion significantly increased, but that of the GC was not altered. Thus, incorporating CLC is an effective technique to cool equine semen, although it is indicated mainly for BC stallions.

New seminal plasma removal method for freezing stallion semen

Seminal plasma removal, an indispensable step in equine semen cryopreservation, is usually done by centrifugation, but this might cause mechanical damage to sperm. A new method for seminal plasma removal from stallion semen, namely a filter composed of a synthetic hydrophilic membrane (Sperm Filter, BotuPharma, Botucatu, Sao Paulo, Brazil), was recently proposed. The objective of this study was to test the use of the Sperm Filter in the removal of seminal plasma before freezing stallion semen. Ejaculates from 31 stallions were divided into two groups and cryopreserved. In group 1 (G1), seminal plasma was removed with the Sperm Filter, and in group 2 (G2), seminal plasma was removed by centrifugation (600×g for 10 minutes). There were no differences (P < 0.05) between G1 and G2 in sperm kinetic parameters or plasma membrane integrity before or after cryopreservation. However, sperm recovery rate was higher (P < 0.05) for G1 versus G2 (mean ± SD, 89.4 ± 7.4% vs. 80.9 ± 5.5%). Therefore, the Sperm Filter was as efficient as centrifugation in removing seminal plasma from the stallion ejaculate. However, filtering was more practical and had significantly fewer sperm lost than the centrifugation technique.

Effect of the Flunixin Meglumine on Pregnancy Rates in an Equine Embryo Transfer Program

ABSTRACT During the equine embryo transfer (ET), manipulation of the recipients cervix can stimulate the release of prostaglandin F2&agr; by the uterine environment. Nonsteroidal anti‐inflammatory drugs such as flunixin meglumine (FM) are frequently used in order to prevent a potential luteolysis. However, despite the reduction of inflammatory reaction and release of prostaglandins, the benefits of FM in pregnancy rates (PRs) of mares submitted to ET are not conclusive, and there is no information about the early pregnancy loss (EPL) rate after FM injection. The objective of this study was to evaluate the effect of FM in the PR and EPL in embryo‐recipient mares. The data from 409 ET from a commercial breeding center were used, which 179 mares formed the control group (CG) and 230 recipients received the treatment of FM 1.1 mg/kg immediately after ET. There was no difference (P > .05) in PRs at 15 days (70.95% in the CG and 75.22% in treated mares) and 60 days (65.92% in CG and 65.22% in FM treated mares). However, there was a trend in the increase of early the pregnancy loss rate in mares that received FM (P = .0852). From the results of the present experiment, FM does not improve the PR in embryo‐recipient mares. HighlightsThe embryonic prostaglandins activate mobility mechanism in the entire uterine lumen, essential to maternal recognition of pregnancy and the antiluteolytic factor distribution.Nonsteroidal anti‐inflammatory drugs such as flunixin meglumine (FM) suppress prostaglandin production and are frequently used in an equine embryo transfer program to prevent a potential luteolysis.Flunixin meglumine does not improve the pregnancy rate in embryo‐recipient mares.Treatment with FM showed a tendency to increase the pregnancy loss.

Fixed-time insemination with frozen semen in mares: is it suitable for poorly fertile stallions?

The purpose of the present study was to compare two protocols for equine frozen semen programs using either postovulation insemination or fixed-time insemination (FT), evaluating both pregnancy rates and intrauterine fluid (IUF) accumulation after artificial insemination with semen obtained from either highly or poorly fertile stallions. Six ejaculates from two stallions (n = 12) were processed. After thawing, semen samples were evaluated by computerized semen analysis. Fifteen mares (30 cycles) were inseminated with frozen semen from highly fertile stallion A, and 14 mares (28 cycles) were inseminated with frozen semen from poorly fertile stallion B. Ovulations were induced with 1 mg (intramuscular) of deslorelin acetate after the observation of a greater than 35 mm follicle and uterine edema. In postovulation insemination group, mares were inseminated once with 800 × 10(6) total sperm in a maximum 6-hour interval after ovulation. In FT group, mares were inseminated twice with 400 × 10(6) total sperm, 24 and 40 hours after induction. Mares were ultrasonographically examined for IUF accumulation 24 hours and for pregnancy diagnosis 14 days after the last insemination. Although IUF accumulation was more evident in mares inseminated once postovulation, pregnancy rates were similar for both protocols, regardless of the stallion, although a significant effect of the stallion was observed. These results indicated that FTs may be used for both highly and poorly fertile stallions as a practical tool to help spreading the use of frozen semen in equine reproduction programs.

Cooling of ejaculated and epididymal stallion sperm

A recuperacao de espermatozoides da cauda do epididimo pode ser a ultima chance para preservacao do germoplasma quando ocorre morte subita ou lesao grave em garanhoes de alto valor genetico. O presente trabalho comparou a viabilidade apos refrigeracao dos espermatozoides do ejaculado (G1), recuperados da cauda do epididimo imediatamente apos a orquiectomia (G2) e recuperados apos armazenamento do epididimo por 24 horas a 5oC (G3). No G1 foram colhidos dois ejaculados. Uma semana apos a colheita dos ejaculados os garanhoes foram submetidos a orquiectomia bilateral e realizada a colheita dos espermatozoides da cauda do epididimo de um testiculo de cada garanhao (G2). O testiculo contralateral permaneceu a 5°C por 24 horas, antes da recuperacao espermatica (G3). A analise das amostras foi realizada imediatamente apos a adicao do meio de refrigeracao, e apos 24 e 48 horas de armazenamento a 5°C. Apos 24 e 48 horas de armazenamento, os espermatozoides do epididimo demonstraram caracteristicas de cinetica maiores que os do ejaculado (P<0.05). Estes resultados indicam que espermatozoides recuperados da cauda do epididimo foram mais resistentes ao processo de refrigeracao, com maiores parâmetros de cinetica espermatica e integridade da membrana plasmatica quando comparados aos espermatozoides do ejaculado.

Pentoxifylline effects on capacitation and fertility of stallion epididymal sperm

The aims of this study were to determinate whether pentoxifylline (PTX) increases the motion parameters of fresh and frozen-thawed equine epididymal spermatozoa, to evaluate the tyrosine phosphorylation of frozen-thawed epididymal sperm in the presence of PTX and to determine whether the PTX-treatment of stallion epididymal sperm prior to freezing improves the fertility response of mares to a reduced number of spermatozoa per insemination dose. Fifty epididymis were flushed with a skim milk based extender with or without PTX. The pre-treatment with PTX enhanced the sperm motility after being harvested (P<0.05); however the freeze-thaw process did not alter the sperm kinematics between control and treated samples (P>0.05). Plasma membrane integrity did not differ between control and PTX group after recovery and after thawing (P>0.05), as observed in tyrosine phosphorylation, which the PTX treatment did not alter the percentage of tail-associated immunofluorescence of cryopreserved epididymal sperm (P>0.05). For the fertility trial, different insemination groups were tested: 800×106 epididymal sperm (C800); 100×106 epididymal sperm (C100); 100×106 epididymal sperm recovered in an extender containing PTX (PTX100). The conception rates for C800; C100 and PTX100 were 68.7% (11/16); 31.5% (5/16) and 50% (8/16), respectively. The conception rate did not differ among groups (P>0.05), however, a low number of animals was used in this study. A trend toward significance (P=0.07) was observed between C800 and C100 groups. In conclusion, PTX has no deleterious effect on sperm motility, viability and capacitation of cryopreserved stallion epididymal sperm. The conventional artificial insemination with 100×106 sperm recovered with PTX ensures acceptable conception rates and maximize the limited number of doses of cryopreserved stallion epididymal sperm.

Effect of cholesterol on plasmatic and acrosomal membrane integrity of cooled equine sperm

The aim of this study was to analyze the follicle and oocytes morphometry from different follicular classes. The ovaries of 17 queens in anestrus were classified into three groups: Young (0–1 year), Adults (1–6 years) and Older (>6 years). The ovaries were fixed in paraformaldehyde 5%, embedding in paraffin and staining with haematoxylin-eosin. For morphological analysis the tissue sections were photographed by microscope (Olympus BX61) and classified as primordial, unilaminar primary, multilaminar primary, secondary and pre-ovulatory follicles. A total of 1039 follicles were measured and the parameters utilized were: diameter (lm), area (lm2) and perimeter (lm). The statistical used were ANOVA and the means were compared by Tukey test and medians using the Kruskal-Wallis test followed by Dunn’s multiple comparisons (p < 0.05). In young queens primordial follicles there were increased in the mean diameter, area and perimeter of follicle (45.16 lm, 1941 lm and 157.24 lm) and oocytes (40.55 lm, 1320.4 lm and 129.90 lm) when compared to adults (Follicles: 41.51 lm, 1652.4 lm e 145.56 lm and in oocytes: 37.57 lm, 1134.3 lm and 120.58 lm). A biphasic pattern of follicle and oocyte growth was observed through linear regression. Before antrum formation, follicle (x) and oocyte (y) size were positively and linearly correlated (y = 0.304x + 25.01, r = 0.72), although after antrum formation a negative correlation were found (y = 0.007x + 98.00, r = 0.00). The queen offers many benefits as a model of ovarian folliculogenesis, and may be useful in preserving of endangered animals. Acknowledgements: FAPESP for financial support.

Effect of cryopreservation on sperm membranes of bad and good freezer stallions

The aim of this study was to analyze the follicle and oocytes morphometry from different follicular classes. The ovaries of 17 queens in anestrus were classified into three groups: Young (0–1 year), Adults (1–6 years) and Older (>6 years). The ovaries were fixed in paraformaldehyde 5%, embedding in paraffin and staining with haematoxylin-eosin. For morphological analysis the tissue sections were photographed by microscope (Olympus BX61) and classified as primordial, unilaminar primary, multilaminar primary, secondary and pre-ovulatory follicles. A total of 1039 follicles were measured and the parameters utilized were: diameter (lm), area (lm2) and perimeter (lm). The statistical used were ANOVA and the means were compared by Tukey test and medians using the Kruskal-Wallis test followed by Dunn’s multiple comparisons (p < 0.05). In young queens primordial follicles there were increased in the mean diameter, area and perimeter of follicle (45.16 lm, 1941 lm and 157.24 lm) and oocytes (40.55 lm, 1320.4 lm and 129.90 lm) when compared to adults (Follicles: 41.51 lm, 1652.4 lm e 145.56 lm and in oocytes: 37.57 lm, 1134.3 lm and 120.58 lm). A biphasic pattern of follicle and oocyte growth was observed through linear regression. Before antrum formation, follicle (x) and oocyte (y) size were positively and linearly correlated (y = 0.304x + 25.01, r = 0.72), although after antrum formation a negative correlation were found (y = 0.007x + 98.00, r = 0.00). The queen offers many benefits as a model of ovarian folliculogenesis, and may be useful in preserving of endangered animals. Acknowledgements: FAPESP for financial support.

Effects of cryoprotectant on viability and fertility of chilled bull semen

The aim of this study was to analyze the follicle and oocytes morphometry from different follicular classes. The ovaries of 17 queens in anestrus were classified into three groups: Young (0–1 year), Adults (1–6 years) and Older (>6 years). The ovaries were fixed in paraformaldehyde 5%, embedding in paraffin and staining with haematoxylin-eosin. For morphological analysis the tissue sections were photographed by microscope (Olympus BX61) and classified as primordial, unilaminar primary, multilaminar primary, secondary and pre-ovulatory follicles. A total of 1039 follicles were measured and the parameters utilized were: diameter (lm), area (lm2) and perimeter (lm). The statistical used were ANOVA and the means were compared by Tukey test and medians using the Kruskal-Wallis test followed by Dunn’s multiple comparisons (p < 0.05). In young queens primordial follicles there were increased in the mean diameter, area and perimeter of follicle (45.16 lm, 1941 lm and 157.24 lm) and oocytes (40.55 lm, 1320.4 lm and 129.90 lm) when compared to adults (Follicles: 41.51 lm, 1652.4 lm e 145.56 lm and in oocytes: 37.57 lm, 1134.3 lm and 120.58 lm). A biphasic pattern of follicle and oocyte growth was observed through linear regression. Before antrum formation, follicle (x) and oocyte (y) size were positively and linearly correlated (y = 0.304x + 25.01, r = 0.72), although after antrum formation a negative correlation were found (y = 0.007x + 98.00, r = 0.00). The queen offers many benefits as a model of ovarian folliculogenesis, and may be useful in preserving of endangered animals. Acknowledgements: FAPESP for financial support.