Chitosan-dextran sulphate nanoparticles for GnRH release in rabbit insemination extenders.

This study was designed to develop chitosan (CS)-dextran sulphate (DS) nanoparticles containing a GnRH analogue and to study their effect on rabbit (Oryctolagus cuniculus) semen quality. Six experimental extenders were tested as follows: (control) Tris-citric acid-glucose (TCG), (1) 0.05% CS-0.05% DS (4:1), (2) 0.1% CS-0.05% DS (4:1), (3) 0.05% CS-0.05% DS (3:1), (4) 0.1% CS-0.05% DS (3:1), (5) 0.1% CS-0.05% DS (2:1). CS and DS were dissolved in TCG medium, and nanoparticles were obtained through magnetic stirring. Rabbit seminal samples were incubated up to 5 hr at 37°C in the extenders, and seminal quality was evaluated. The entrapment efficiency was 40%-50%. After 5 hr at 37°C, a 20% of the hormone was released. Results showed that the presence of CS-DS nanoparticles did not affect rabbit semen motility, viability and membrane functionality; however, acrosome integrity was significantly higher versus control (p < .001).

Can the Genetic Origin Affect Rabbit Seminal Plasma Protein Profile along the Year?

The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240 kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI-TOF/TOF or LC-MS/MS analysis and were the following ones: FAM115E-like (220, 113 and 59 kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72 kDa), annexin A5 (32 kDa), lipocalin allergen Ory c 4 precursor (19 kDa), and haemoglobin subunit zetalike (13 kDa) between genetic lines and FAM115E-like (113 kDa), haemoglobin subunit zetalike (13 kDa) and ß-nerve growth factor (12 kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies.

Porcine oocyte vitrification in optimized low toxicity solution with open pulled straws.

One of the greatest challenges for reproductive cryobiologists today is to develop an efficient cryopreservation method for human and domestic animal oocytes. The objective of the present study was to optimize a low toxicity solution called VM3 to vitrify porcine oocytes using an open pulled straw (OPS) device and to evaluate the effects on viability, chromosomal organization and cortical granules distribution. Two experiments were conducted in this study. Firstly, we determined the minimum concentration of cryoprotectant present in the VM3 solution required (7.6 M) for vitrification using an OPS device. The appearance of opacity was observed when using a cooling solution at -196°C; no observable opacity was noted as vitrification. In addition, the ultrastructure of oocytes in VM3 or VM3 optimized solution was examined using cryo-scanning electron microscopy. The minimum total cryoprotectant concentration present in VM3 solution necessary for apparent vitrification was 5.6 M when combined with use of an OPS device. Use of both vitrification solutions showed a characteristic plasticized surface. In the second experiment, the relative cytotoxicity of vitrification solutions (VM3 and VM3 optimized) was studied. Oocyte viability, chromosomal organization and the cortical granules distribution were assessed by fluorescent stain. After warming, oocyte survival rate was similar to that of fresh oocytes. The vitrification process significantly reduced correct chromosomal organization and cortical granules distribution rates compared with the fresh oocytes group. However, correct chromosomal organization and cortical granules distribution rates did not differ among oocytes placed in different vitrification solutions. In conclusion, our data demonstrated that the VM3 solution can be optimized and that reduction in concentration to 5.6 M enabled vitrification of oocytes with an OPS device, however use of the VM3 optimised solution had no beneficial effect on vitrification of porcine oocytes.

Effect of cytochalasin B pre-treatment of in vitro matured porcine oocytes before vitrification.

The study tested the efficacy of pre-treating mature porcine oocytes with cytochalasin B before vitrification by the open pulled straw method (OPS) in a low toxicity solution containing ice blockers. The effects of pre-treating the oocytes with 7.5 micrograms per ml cytochalasin B before vitrification on membrane integrity, chromosome organisation and cortical granule distribution were evaluated. When oocytes pre-treated with cytochalasin B before vitrification were compared with control oocytes, similar membrane integrity was observed. In contrast, when both vitrified oocytes groups (treated and untreated with cytochalasin B) were compared with fresh oocytes, significantly lower proportions of oocytes with normal chromosomes aligned regularly on the metaphase plate and peripheral cortical granule distribution were observed. The percentages of oocytes with normal chromosomes aligned regularly on the metaphase plate were similar between those treated or untreated with cytochalasin B before vitrification. Similar results were found for normal cortical granules distribution. Irrespective of previous cytochalasin B exposure, vitrification gave rise to higher abnormal cortical granule distribution percentages. Cytochalasin B pretreatment of oocytes before vitrification does not help to reduce the damage induced by the cryopreservation process of porcine oocytes.

Protection of GnRH analogue by chitosan-dextran sulfate nanoparticles for intravaginal application in rabbit artificial insemination.

The present study was designed to prove new rabbit insemination extenders containing aminopeptidase inhibitors (AMIs) with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. In addition, different hormone concentrations were tested in these extenders, evaluating their in vivo effect on rabbit reproductive performance after artificial insemination. A total of 911 females were inseminated with semen diluted with the four experimental extenders (C4 group: 4 µg buserelin/doe in control medium (Tris-citric acid-glucose supplemented with bestatin 10 µM and EDTA 20 mM), C5 group: 5 µg of buserelin/doe in control medium, Q4 group: 4 µg of buserelin/doe into CS-DS nanoparticles in control medium, Q5 group: 5 µg of busereline/doe into CS-DS nanoparticles in control medium). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 versus 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied (P > 0.05). We conclude that the CS-DS nanoparticles prepared by a coacervation process as carrier for buserelin acetate allows reducing the concentration of hormone used in extenders supplemented with bestatin and EDTA without affecting the fertility and prolificacy of rabbit females.

Insemination extender supplementation with bestatin and EDTA has no effect on rabbit reproductive performance.

The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, ß nerve growth factor (ß-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as ß-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and ß-NGF, they could be used to develop new extenders with less hormone concentration.

Does the inclusion of protease inhibitors in the insemination extender affect rabbit reproductive performance?

The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of aminopeptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 ± 0.26 and 9.3 ± 0.23 vs. 8.2 ± 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size.