Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski's horse (Equus ferus przewalskii)

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski's horse (Equus ferns przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide IMF] and dimethylformamide [DMF]) to Przewalski's horse spermatozoa during liquid storage at 4 C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski's versus domestic horse. When Przewalski's horse spermatozoa were incubated at 4 C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P < 0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P> 0.05) between the Przewalski's (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose-EDTA-glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondria] membrane potential were 42.4%, 21.8%, 88.7% and 25.4 CN (CN = mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski's and 49.3%, 24.6%, 88.9% and 25.8 CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P> 0.05) among treatments across species, mitochondrial membrane potential was higher (P< 0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski's stallion sperm expressed higher (P < 0.05) post-thaw total motility in BOTU and SM compared to EQ whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski's stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski's and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski's horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF. Published by Elsevier Inc.

The influence of insemination dose on pregnancy per fixed-time artificial insemination in beef cows is affected by semen extender

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of Botu-bov and Tris as Freezing Extenders of Buffalo Sperm Recovered from Epididymal Cauda

Valuable genetic material can be preserved by the cryopreservation of epididymal sperm. This study evaluated the viability of pre-freezing and post-thawed sperm samples recovered from the epididymal cauda of buffaloes. Epididymides from eight Murrah buffaloes with 18 months of age were used. Semen samples were diluted in two different freezing extenders: Botu-bov (BB) and Tris (TRIS). Immediately after slaughter, both testicles from each animal were collected and transported at 4 degrees C for six hours interval. In laboratory, the removed epididymides were flushed to obtain sperm and the fractions were diluted in both freezing extenders (BB and TRIS). Semen doses were analyzed before and after frozen at -196 degrees C. BB and TRIS pre-freezing results were 38.54 +/- 22.33%(b) and 14.17 +/- 12.78%(a) for total motility (TM), 25.00 +/- 16.12(a) and 9.44 +/- 9.11(a) for progressive motility (PM), 7.21 +/- 0.98(a) and 5.09 +/- 2.65(a) for percentage of rapid cells (RAP), 91.08 +/- 12.53(b) and 63.33 +/- 31.47(a) for velocity of trajectory (VAP), 73.54 +/- 20.17(b) and 49.50 +/- 9.11(a) for linear progressive velocity (VSL), 172.21 +/- 24.55(a) and 116.94 +/- 59.48(a) for curvilinear velocity (VCL), respectively (P < 0.05). BB and TRIS post-thawing results were 42.25 +/- 21.50(b) and 17.62 +/- 19.46(a) for TM, 27.25 +/- 24.86(a) and 18.00 +/- 13.68(a) for PM, 7.35 +/- 0.98(a) and 6.26 +/- 1.13(a) for RAP, 91.42 +/- 16.86(a) and 75.96 +/- 13.17(a) for VAP, 67.96 +/- 12.13(a) and 60.04 +/- 10.42(a) for VSL, 177.54 +/- 23.53(b) and 141.29 +/- 24.97a for VCL, respectively (P < 0.05). The sperm recovered from the epididymal cauda, after 6 h storage of epididymides at 5 degrees C ensures sperm preservation demonstrating that the diluent Botu-bov had higher total motility both pre-and post-freezing when compared with TRIS. Additionally, the sperm frozen with the diluent Botu-bov showed higher values of VSL at post-thawing. These findings may reflect in improvement of conception rates.

Adding Motility Stimulants to Improve Freezing of Buffalo Sperm Recovered from Epididymal Cauda

The cryopreservation of epididymal sperm is important to preserve genetic material from valuable buffalo bulls. This study evaluated the viability of post-thawed sperm samples recovered from the epididymal cauda adding motility inductors. For that, were used epididymides from eight Murrah buffaloes with 18 months of age. Semen samples were submitted to three different conditions: (CT - control) without adding medium, (SPERM) adding Sperm Talp medium, and (FERT) adding Fert Talp medium. Immediately after slaughter, both testicles from each animal were collected and transported at 4 degrees C at maximum six hours interval. In laboratory, the removed epididymides was flushed to obtain sperm and diluted in the freezing extender. Each buffalo sperm were divided and fractions were submitted to all conditions (CT, SPERM and FERT). Semen doses were frozen at -196 degrees C. CT, SPERM and FERT post-thawing results were 13.63 +/- 8.91, 38.77 +/- 8.91 and 42.83 +/- 8.91 for total motility, 7.30 +/- 8.74, 24.87 +/- 8.74 and 29.70 +/- 8.74 for progressive motility, 6.04 +/- 0.92, 6.74 +/- 0.92 and 6.93 +/- 0.92 for percentage of rapid cells (P < 0.05). In conclusion, diluted semen supplementation with Sperm or Fert talp increases the motility of cauda epididymal sperm of buffalo bulls.

Effect of platelet-rich plasma on bone healing of fresh frozen bone allograft in mandibular defects: a histomorphometric study in dogs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Uso de sêmen liofilizado de gatos domésticos (Felis silvestris catus) para produção in vitro de embriões através da técnica de injeção intracitoplasmática de espermatozóide (ICSI)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Indução a espermiação e criopreservação espermática de Brycon cephalus (Gunther, 1869) (teleostei: characidae)

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Effect of platelet-rich plasma on the healing of mandibular defects treated with fresh frozen bone allograft: a radiographic study in dogs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Morfometria e compactação da cromatina espermática de touros Nelore de acordo com a idade e sua influência na produção de embriões in vitro

Pós-graduação em Medicina Veterinária - FCAV

Congelação de células espermáticas provenientes de epidídimo de gatos domésticos contendo antioxidante no meio diluidor

Pós-graduação em Medicina Veterinária - FCAV

Remodeling of cortical and corticocancellous fresh-frozen allogeneic block bone grafts - a radiographic and histomorphometric comparison to autologous bone grafts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

Control Methods and Evaluation of Bacterial Growth on Fresh and Cooled Stallion Semen

The penis and prepuce of the stallion have a high bacterial load on its surface, forming a natural microbial flora that contaminates the semen during ejaculation. Bacterial growth in semen may cause a decline on sperm quality, viability, and fertility and predisposes the occurrence of endometritis in inseminated mares. Thus, the aim of this study was to evaluate the effect of penile wash before semen collection, the addition of different commercial skim milk-based extenders containing antibiotics (BotuSemen and INRA96), and the removal of seminal plasma by filtration on the quality, viability, and bacterial proliferation on fresh and cooled stallion semen. Animals that were never submitted to penile wash before semen collection tended to have lower bacterial contamination in the ejaculate. Semen samples extended in BotuSemen showed superiority in total motility, progressive motility, average path velocity, and rapid sperm and lower bacterial contamination in relation to semen samples extended in INRA96 after 24 hours of cooling. No difference was found in these parameters between the storage temperatures (5 degrees C and 15 degrees C). Furthermore, the removal of seminal plasma by filtration reduced the bacterial load in semen after cooling. In conclusion, the penile wash before semen collection tended to reduce the bacterial growth in fresh semen. The use of a semen extender with appropriate antibiotics and removal of seminal plasma by filtration were effective in reducing the bacterial contamination and preserved the quality of cooled stallion semen. (C) 2015 Elsevier Inc. All rights reserved.