Recombinant TrxAFNIIx4His6 improves post-thaw motility of ram sperm measured by a sperm motility tracker software.

The aim of the present study was to evaluate a freezing extender supplemented with recombinant TrxAFNIIx4His6, a reported decapacitating factor. Semen samples were diluted in tris-egg yolk medium with 0, 1.5 µM and 3.0 µM of TrxAFNIIx4His6. Computer-assisted sperm motility tracking and subpopulations evaluation showed that addition of TrxAFNIIx4His6 improved post-thaw total and progressive motility at both concentrations evaluated. TrxAFNIIx4His6 increased the sperm subpopulation with the highest progressiveness and great velocity and decreased the subpopulation of poorly motile and almost non-progressive sperm. Incorporation of TrxAFNIIx4His6 to freezing extender shows potential for the development of cryoprotection media which may lead to improved fertility after artificial insemination.

Desmosterol Incorporation Into Ram Sperm Membrane Before Cryopreservation Improves in vitro and in vivo Fertility.

Pregnancy rates in ewes are markedly low after cervical insemination with frozen-thawed sperm. Sensitivity of ram sperm to freeze-thawing is related to the lipid composition of the membrane, particularly to its low sterol content. Recently, we proved that sterol content of ram sperm can be increased by treatment with methyl-ß-cyclodextrin-sterol complexes and we provided mechanistic based evidence on the differential behavior of cholesterol and desmosterol in the ram sperm membrane. In the present study, we evaluated the role of increasing cholesterol and desmosterol content of ram sperm before cryopreservation, on the extent and distribution of sterols, cryocapacitation status, acrosome integrity, DNA damage associated with apoptosis and fertility competence in vitro and in vivo of post-thawed sperm. After freeze-thawing, similar levels of sterol content were evidenced in control sperm cells and in those pre-incubated with either cholesterol or desmosterol. Still, moderately higher levels of sterols were registered in treated sperm compared to the control, indicating no physiological excess of sterols after thawing or sterol losses that exceed the control. Live cell imaging of fluorescent cholesterol evidenced the presence of sperm sub-populations differentially affected by freeze-thawing. Similar unimodal frequency profiles were observed between sterol-enriched groups, while the control exhibited a sub-population of sperm compatible with low sterol content. Tyrosine phosphorylation was significantly lower when ram sperm incorporated cholesterol compared to the control. No difference in this capacitation parameter was found between the latter and desmosterol-enriched sperm. The percentage of sperm with damaged acrosomes post-thawing, assessed by a fluorescent lectin, was reduced in sperm that incorporated sterols before freezing, irrespective of the sterol class. These results suggest that sterols exert a stabilizing effect on the acrosome. No differences were found in levels of apoptotic DNA fragmentation among experimental groups. As to fertility trials, desmosterol-enriched sperm gave rise to higher rates of in vitro activated oocytes by heterologous fertilization and to significantly lower pregnancy loss in vivo. Our research provides new insights on sterol incorporation into ram sperm prior to cryopreservation, in particular on the additional benefit of incorporating desmosterol as a strategy to improve fertility outcome.

Cholesterol and desmosterol incorporation into ram sperm membrane before cryopreservation: Effects on membrane biophysical properties and sperm quality.

Ram sperm are particularly sensitive to freeze-thawing mainly due to their lipid composition, limiting their use in artificial insemination programs. We evaluated the extent of cholesterol and desmosterol incorporation into ram sperm through incubation with increasing concentrations of methyl-ß-cyclodextrin (MßCD)-sterol complexes, and its effect on membrane biophysical properties, membrane lateral organization and cryopreservation outcome. Sterols were effectively incorporated into the sperm membrane at 10 and 25 mM MßCD-sterols, similarly increasing membrane lipid order at physiological temperature and during temperature decrease. Differential ordering effect of sterols in ternary-mixture model membranes revealed a reduced tendency of desmosterol of segregating into ordered domains. Live cell imaging of fluorescent cholesterol showed sterol incorporation and evidenced the presence of sperm sub-populations compatible with different sterol contents and a high concentration of sterol rich-ordered domains mainly at the acrosome plasma membrane. Lateral organization of the plasma membrane, assessed by identification of GM1-related rafts, was preserved after sterol incorporation except when high levels of sterols (25 mM MßCD-desmosterol) were incorporated. Ram sperm incubation with 10 mM MßCD-sterols prior to cryopreservation in a cholesterol-free extender improved sperm quality parameters after cooling and freezing. While treatment with 10 mM MßCD-cholesterol increased sperm motility, membrane integrity and tolerance to osmotic stress after thawing, incorporation of desmosterol increased the ability of ram sperm to overcome osmotic stress. Our research provides evidence on the effective incorporation and biophysical behavior of cholesterol and desmosterol in ram sperm membranes and on their consequences in improving functional parameters of sperm after temperature decrease and freezing.

Extenders modify the seminal plasma ability to minimize freeze-thaw damage on ram sperm.

Seminal plasma (SP) proteins interact with sperm plasma membrane (PM) modulating its functionality. It has been shown that SP proteins can reverse the damage caused by freeze-thaw; however in these studies, SP has been added to washed sperm (i.e., cells depleted from homologous SP and extender). The aim of the current study was to assess whether the egg yolk-based extender (EY) modifies SP ability to ameliorate sperm parameters in frozen-thawed ram spermatozoa. Ejaculates were diluted in EY or soybean lecithin-based extender (SL) and evaluated before and after freezing to measure the cell damage according to the extender. Even when all classical parameters decreased after freezing, as expected (p < .05), there was no effect of the extender. SP treatment was applied after freeze-thaw. Sperm were incubated with SP (20% v/v) in the presence of either EY or SL, and sperm parameters were assessed after thawing compared with the same treatments after Percoll sperm selection (washed). Treatments with 20% SP improved sperm total and progressive motility compared with controls regardless of washing and extender (p < .05); however, washed sperm showed higher percentage of total sperm motility compared with those unwashed (p < .05). Moreover, treatment with 20% SP showed significantly higher percentages of PM integrity, sperm with intact acrosomes, integrity of chromatin and non-capacitated sperm in samples diluted with EY when washed before treatment compared with the other conditions (p < .05). It was concluded that the presence of the extenders and particularly egg yolk alters the SP capacity to reduce the cryodamage.

Cryopreservation and egg yolk extender components modify the interaction between seminal plasma proteins and the sperm surface.

It is known that the addition of seminal plasma (SP) or SP proteins either before freezing or post thawing show contradictory results on sperm quality and fertility due to the interference between SP and the extender. Thus, the aim of this study was to determine whether egg yolk (EY) interferes with SP ability to protect the functionality and fertility of ram sperm during freeze-thawing by modifying the interaction between seminal plasma proteins and the sperm plasma membrane. Ejaculated or epididymal ram sperm collected during the breeding season were incubated with SP in the presence or absence of EY or soybean lecithin-based extenders before cryopreservation. No significant differences were observed after thawing in sperm quality (total and progressive sperm motility, membrane integrity, plasma membrane functionality, percentage of non-capacitated sperm) between the extenders, either in presence or absence of seminal plasma (P ≥ 0.05). The amount of proteins retained by the sperm surface normalized to number of cells was diminished after freeze-thawing compared to their fresh counterparts for all the treatments (P < 0.05), demonstrating that cryopreservation weakens the interaction between external proteins and the sperm surface. The electrophoretic analysis of sperm-bound proteins showed that the retention of several SP peptides onto the sperm surface (based on densitometry estimation) was affected by the presence of the diluents on both ejaculated and epididymal sperm (P < 0.05). Moreover, variation was observed in the protein pattern after thawing compared to the corresponding fresh samples, suggesting that freezing affects surface protein profile. Pregnancy rate after artificial insemination at fixed time was higher (P < 0.05) for samples treated with reconstituted with heterologous SP compared to those supplemented with 20% additional seminal plasma or control samples despite the presence of EY. In conclusion, both freeze-thawing and EY components affected the interaction among seminal plasma proteins and the sperm surface, although these changes were not reflected on different sperm quality parameters under our experimental conditions. In vivo fertility of sperm reconstituted with exogenous SP before freezing was improved even in the presence of EY components considering an optimal ratio SP:sperm.

Recombinant peptide reverses cryo-capacitation in ram sperm and improves in vitro fertilization.

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 µM), TrxA-FNIIx4-His6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ±â€¯2.95; 40.13 ±â€¯11.82 for 0.3 µM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability.

Seminal plasma proteins modify the distribution of sperm subpopulations in cryopreserved semen of rams with lesser fertility.

Any physiological mechanism involved in sperm selection and semen improvement has effects on heterogeneous sperm populations. This is mainly due to the fact that sperm populations within a single ejaculate have considerable heterogeneity for many variables, such as motility which is meaningful in terms of understanding how some sperm cells possess fertility advantages as compared with other cells. In the present research, initially there was a multivariate and clustering analysis used to assess sperm motility data from cryopreserved ram semen to identify subpopulations and compare the distribution of these clusters between rams with lesser and greater fertility. There were four classifications made of sperm subpopulations (clusters): CL1 fast/linear/progressive sperm; CL2 fast/non-linear sperm; CL3 very fast/linear sperm with vigorous beating and CL4 slow/non-linear sperm. Rams with greater fertility had a lesser proportion of sperm considered as "hyperactivated" (CL2) and a greater proportion of slow and non-linear sperm (CL4) than sperm of rams with lesser fertility. In addition, the effects were assessed for the capacity of seminal plasma (SP) and interacting SP proteins (iSPP) that were present during different seasons of the year to improve the distribution of sperm within subpopulations of semen from rams with lesser fertility. The iSPP and SP were obtained by artificial vagina (AV) and electroejaculation (EE) during breeding and non-breeding seasons and added to thawed semen. All the aggregates had a significant effect on the distribution of sperm subpopulations and effects differed among seasons of the year and depending on collection method used. Even though, future studies are needed to assess the contribution of each subpopulation on ram sperm fertility, it is important that a multivariate analysis be used to evaluate the effect of a treatment on sperm quality variables.

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat ß-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.

Seminal plasma proteins interacting with sperm surface revert capacitation indicators in frozen-thawed ram sperm.

This study was conducted to evaluate the effects of interacting seminal plasma proteins (iSPP) obtained by AV or EE on frozen-thawed ram sperm in order to test the hypothesis whether this fraction could be sufficient to emulate the effect of complete seminal plasma (SP). Additionally, we evaluated whether these proteins have a differential effect between spermatozoa from high and low fertility rams and between breeding and non-breeding seasons. We assessed sperm motility, quality parameters (intracellular reactive oxygen species, membrane fluidity, plasma membrane permeability and mitochondrial activity) and capacitation status. The main findings from this work were: i) iSPP had no effect on sperm motility, whereas SP (AV or EE) addition produced the highest values of total motility (74.13±2.99 and 72.27±2.99 for AV and EE, respectively) and progressive motility (64.97±2.64 and 63.73±2.64 for AV and EE, respectively); ii) iSPP had no effect on sperm quality parameters (p>0.05), but whole SP improved all parameters evaluated. Moreover, SP collected by AV yielded significantly higher viability (44.60±2.87) and sperm with stable plasma membrane (44.56±2.49) comparing with the addition of SP collected by EE (35.80±2.47 and 36.67±1.71, respectively); iii) iSPP and SP collected by EE, but not by AV, reverted molecular signals of capacitation as protein tyrosine phosphorylation caused by freezing temperatures; iv) there were no effects of fertility or season in sperm quality parameters evaluated. This study demonstrated that, although the iSPP have a clear decapacitating effect, including the ability to revert cryo-capacitation indicators, they are not sufficient to emulate the effects of complete SP regarding sperm functional parameters.

Hematology and serum biochemistry of free-ranging nutria (Myocastor coypus).

Information on reference blood values in the literature is lacking for many wild rodents. In this study, comprehensive reference intervals (RIs) for a wide range of analytes from 101 healthy free-ranging nutria were determined. Animals were captured in Buenos Aires, Argentina (37degrees 50'S, 57 degrees 34'W), and southward (38 degrees 60'S, 58 degrees 23'W), encompassing major biotopes of agricultural pampas with dunes and grassland steppes on the east coast. Traps were set at locations with high-density nutria populations (i.e., those areas that showed signs of movement, territorial marking, or feeding activities). Although the small sample size limits the interpretation of these findings, RIs were determined by a robust method using the central 95th percentile. In nutria, the RI range varied greatly for the leukocyte differentials, with mature neutrophils: 3,907-5,544/mmicrol for females and 3,744-5,900/microl for males; band neutrophils: 0-10/ll for females and 3-18/microl for males; lymphocytes: 4,213 5,940/microl for both sexes combined; monocytes: 165-402/microl for both sexes combined; eosinophils: 13-91/microl for females and 108-165/microl for males; and basophils: 0-87/microl for both sexes combined. Platelet concentration was 543-727 x 10(9)/L for both sexes combined. There was also a wide RI range for biochemistry values for some enzymes, such as alkaline phosphatase: 200-399 IU/L for both sexes combined; cholinesterase: 762-1,407 IU/L for females and 763-1,284 IU/L for males; creatine kinase: 182-552 IU/L for females and 162-451 IU/L for males; amylase: 853-1,865 IU/L for females and 779-1,293 IU/L for males; and glucose concentration 120.2-180.6 mg/dl for both sexes combined. Conversely, there was not a wide pooled RI range for calcium: 7.0-11.2 mg/dl; phosphorous: 6.1-9.3 mg/dl; sodium: 133.0-159.0 mEq/L; potassium: 3.0-8.2 mEq/L; chloride: 101.4-143.0 mEq/L; and urea: 11.3-36.8 mg/dl. The red blood cell indices had a narrow range, with mean corpuscular volume: 84.0 -102.5 fl and mean corpuscular hemoglobin concentration: 18.2-28.8 g/dl, and which was most likely due to strict physiologic controls. The results from this study were similar to those previously reported for farmed nutria.