Detection of the Matrix Metalloproteinases MMP‐2 and MMP‐9 and Tissue Inhibitors of Metalloproteinases TIMP‐1 and TIMP‐2 in Llama (Lama glama) Oviduct

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in several reproductive events like oocyte–spermatozoa interaction and semen liquefaction. In order to study their role in the llama oviductal reproductive process, MMP activity in oviductal fluid (OF) was assayed. Considering that llama genome sequences are partially known, a strategy to procure cDNA sequences of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 was designed. Afterwards, their expression patterns in the different llama oviductal segments were assayed. Gelatine zymograms detected 62 and 94 kDa protease activities that matched MMP‐2 and pro‐MMP‐9, respectively. Expression pattern analysis showed that MMP and TIMP mRNAs were present in ampulla, isthmus, utero‐tubal junction (UTJ) and papilla. Altogether, these findings support the argument that MMPs/TIMPs are produced in the oviduct and secreted into the oviductal lumen. Our results encourage further studies to elucidate the role of these proteins in reproductive oviductal events.     

Immunohistochemical study of matrix metalloproteinases‐2 and ‐9, macrophage inflammatory protein‐2 and tissue inhibitors of matrix metalloproteinases‐1 and ‐2 in normal,purulonecrotic and fungal infected equine corneas

Objective Determine the effects of matrix metalloproteinases (MMPs)‐2, ‐9, macrophage inflammatory protein‐2 (MIP‐2), tissue inhibitors of matrix metalloproteinase (TIMP)‐1 and ‐2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. Procedure Paraffin‐embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP‐2, ‐9, MIP‐2, TIMP‐1 and ‐2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 proteins. Results Matrix metalloproteinases‐2, ‐9, MIP‐2, TIMP‐1 and ‐2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP‐2 and MIP‐2, MMP‐9 and MIP‐2, and MMP‐9 and TIMP‐1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP‐9 and MIP‐2, MMP‐9 and TIMP‐2, MIP‐2 and TIMP‐1, and MIP‐2 and TIMP‐2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. Conclusion Increased immunoreactivity of MMP‐2 and ‐9 in FA and PN samples is indirectly related to MIP‐2 through its role in neutrophil chemo‐attraction. Tissue inhibitors of matrix metalloproteinase‐1 and TIMP‐2 are up‐regulated in equine purulonecrotic and fungal keratitis secondary to MMP‐2 and MMP‐9 expression. The correlation between MMPs ‐2 and ‐9, MIP‐2, TIMPs ‐1 and ‐2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.     

Effect of Chilling Duration on Post‐Thaw Characteristics of Sperm from the North American bison (Bison bison)

The objective of this study was to determine the duration for which sperm from the North American bison (Bison bison) could be chilled prior to being cryopreserved, without compromising post‐ thaw sperm quality. This would permit transport of samples collected remotely, to the laboratory (at 4°C) for cryopreservation. Epididymal sperm from plains bison (n = 11) and ejaculated sperm from wood bison (n = 3) were collected, extended and held at 4°C for extended periods of time. At intervals, an aliquot was cryopreserved. Post‐thaw sperm motion characteristics were evaluated by computer assisted sperm analysis. Representative plains bison sperm samples (n = 3) were evaluated for their in vitro fertilizing ability in a heterologous system using bovine oocytes. There was no statistical difference in total and progressive motility of plains bison epididymal sperm when cryopreserved after chilling for 24, 48 or 72 h. For wood bison ejaculated sperm, there was no difference in total and progressive motility for sperm cryopreserved following 24 or 48 h of chilling. However, one of the three bulls showed significantly poorer fertilization (based on cleavage rate) with sperm chilled for 72 compared to 24 and 48 h prior to freezing. In conclusion, plains bison epididymal sperm can be chilled for 72 h and wood bison ejaculated sperm can be chilled for at least 48 h prior to cryopreservation without compromising post‐thaw sperm motility, while heterologous in vitro fertilization (IVF) assay indicated a between‐bull variation in the in vitro fertilizing ability of sperm chilled for an extended duration before cryopreservation.     

VEGF and MMP‐9: biomarkers for canine lymphoma

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF‐β) stimulates VEGF and MMPs production. VEGF and TGF‐β plasma levels were tested by ELISA, MMP‐2 and MMP‐9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act‐MMP‐9 (P < 0.01) and VEGF (P < 0.05), and lower TGF‐β than controls, and a positive correlation between act‐MMP‐9 and VEGF (P < 0.001). Act‐MMP‐9 and VEGF were significantly higher in T‐cell lymphomas, and in stage V compared with stages III–IV disease, regardless of immunophenotype. VEGF was higher in high‐grade compared with low‐grade T‐cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act‐MMP‐9 and VEGF decreased in B‐cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.     

Effects of exogenous lactoferrin on characteristics and functions of bovine epididymal,ejaculated and frozen-thawed sperm

The purpose of this study was to investigate effects of addition of lactoferrin on characteristics and functions of bovine epididymal, ejaculated, and frozen-thawed sperm. The addition of lactoferrin was significantly (p < .05) effective on increasing values of progressive motility, straightness, and linearity in caput epididymal sperm and values of motility in cauda epididymal sperm. When ejaculated sperm were incubated in capacitation medium, percentages of motile and progressively motile sperm decreased largely within the first period of 30 min, followed by only minor changes. However, the addition of lactoferrin significantly lessened the early decreases of these parameters and additionally promoted capacitation-dependent changes of chlortetracycline staining patterns (from F pattern to B pattern). In other experiments, when ejaculated sperm were exposed to oxidative stress with 100-µM H₂O₂, the addition of lactoferrin partially protected them from dysfunction of flagellar movement and loss of progressive movement. In final experiments with frozen-thawed samples incubated in the capacitation medium, the addition of lactoferrin effectively survived dying sperm and suppressed occurrence of sperm agglutination. These results may suggest biological and biotechnological potentials of lactoferrin for modulation of bovine sperm viability, motility, capacitation state, and preservation in vitro.     

Morphological indices for canine spermatozoa based on the World Health Organization laboratory manual for human semen

Attempting to contribute to the development of a more objective morphological evaluation of dog spermatozoa, in this study the indices of multiple sperm defects (multiple abnormalities index [MAI]; teratozoospermic index [TZI]; sperm deformity index [SDI]) were calculated following the World Health Organization (WHO) guidelines. In Experiment I, the concordance of MAI, TZI and SDI with the proportions of morphologically normal spermatozoa (MNS) was evaluated in fresh ejaculated spermatozoa (dogs = 47). In Experiment II, the potential role of indices as prognostic values was assessed in spermatozoa of different origin and treatment (fresh ejaculated: n = 6; fresh epididymal: n = 6; frozen‐thawed ejaculated: n = 6) by their correlation with different semen parameters (motility, membrane integrity and acrosome status) and with an in vitro sperm function test. Samples with different proportions of MNS showed different values of SDI, the index that better represented the decline of sperm morphology in both fresh and frozen‐thawed samples (Exp. I and II; p < 0.05). No correlations between indices and semen parameters were observed (Exp. II), but when samples were evaluated collectively, negative correlations (SDI‐motility, p = 0.01; SDI‐acrosome integrity, p = 0.002) were found. Including all the defects of each spermatozoon, SDI might be a useful index during morphological analysis and better discriminates the increase in multiple defects. A more objective morphological evaluation for dog spermatozoa was achieved by the WHO method, and in vitro tests allowed to elucidate the validity of SDI as prognostic indicator of in vitro fertilizing potential.     

Effects of supplemental conjugated linoleic acids (CLA) on fresh and post‐thaw sperm quality of Holstein bulls

This study was designed to investigate the effects of feeding‐protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top‐dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post‐thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer‐assisted), viability (Eosin–Nigrosin), membrane integrity (hypo‐osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p < .05) in the CLA‐fed group compared to control group. Also, in CLA‐fed group, the proportion of post‐thaw spermatozoa with abnormal morphology at week 10 of trial was significantly higher in CLA than control group (p < .05). Progressive motility tended to be increased in the CLA‐fed group, although dietary supplementation did not affect other CASA parameters or viability in fresh and frozen‐thawed sperm. In this study, CLA supplementation had little positive effect on fresh or post‐thaw sperm quality of Holstein bulls.     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

Effects of two freezing methods and two cryopreservation media on post‐thaw quality of stallion spermatozoa

Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio^®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio^® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio^® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio^® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio^® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio^® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.     

Tris–Egg Yolk–Glycerol (TEY) Extender Developed for Freezing Dog Semen is a Good Option to Cryopreserve Bovine Epididymal Sperm Cells

Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed^® or a Tris–egg yolk (TEY)‐based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post‐thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed^®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed^®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post‐thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.     

Influence of different anaesthetic protocols over the sperm quality on the fresh,chilled (4°C) and frozen‐thawed epididymal sperm samples in domestic dogs

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine–dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50–100 × 10⁶ spermatozoa ml^?1, 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine–dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Semen quality improvement in boars fed with supplemental wolfberry (Lycium barbarum)

Wolfberry is well known for its health benefits in Asian countries. This study consisted of two experiments. In Experiment 1, nine boars were provided 40 g dried wolfberry per 100 kg body weight per day in addition to regular feed for 160 days (divided into 40 days phases: I, II, III, and IV) under step‐down air temperature conditions. Controls (n = 9) were fed regular feed only. Significant (p < .05 or p < .01) or slight improvements in sperm progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate were observed in the wolfberry group during phases II and III. No differences were observed in semen volume. After combining the data from phases II ~ IV, significant improvements were detected in all aforementioned traits (p < .05 or p < .01), except semen volume. In Experiment 2, the wolfberry group (n = 5) was fed wolfberry for 90 days and exhibited significantly reduced head, tail, and total abnormality rates (p < .05 or p < .01) in both fresh semen and semen stored for 72 hr at 17°C compared to the control group (n = 5). SOD activity also significantly increased in this group of boars. Collectively, the findings of this study suggest that wolfberry has a positive effect on boar semen quality.     

Effect of Post‐Thaw Addition of Seminal Plasma on Motility,Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen‐thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post‐thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP‐αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post‐thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post‐thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.     

Intracellular calcium chelating agent (BAPTA‐AM) aids stallion semen cooling and freezing–thawing

This study aimed to investigate the effects of different concentrations of 1,2‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N0 N0‐tetraacetic acid, tetra‐acetoxymethyl ester (BAPTA‐AM), an intracellular calcium chelating agent, on stallion semen cooling and freezing–thawing. After collection, semen was extended (1:1 v/v) on a skim milk‐based extender, centrifuged and resuspended at 400 million/ml into cooling or freezing extenders containing 0, 5, 25, 50, 100 and 200 μΜ BAPTA‐AM. Motility parameters were assessed after cooling in Equitainer at 5°C for 12, 24, 48, 72 and 120 hr and after freezing–thawing. In addition, mitochondrial membrane potential, intracellular ATP, reactive oxygen species and malondialdehyde concentrations were measured in cryopreserved‐thawed semen. Cooled stored (48 hr) semen containing 50 μΜ BAPTA‐AM and control extender (0 μΜ BAPTA‐AM) was used to assess fertility. Inclusion of 50 μΜ BAPTA‐AM resulted in superior sperm motility parameters during cooled storage when compared to other groups (p < 0.05). Furthermore, semen cryopreserved in extender containing 50 μΜ BAPTA‐AM showed increased intracellular ATP and mitochondrial membrane potential, whereas reactive oxygen species and malondialdehyde were increased after thawing for all groups (p < 0.05). Addition of 50 μΜ BAPTA‐AM to cooling extender resulted in similar pregnancy rates to the control group (75% vs. 73.6%, respectively; p > 0.05). In conclusion, the addition of BAPTA‐AM to semen extenders aided stallion semen cryopreservation in a dose‐dependent manner. Furthermore, the cooling extender supplemented with 50 μΜ BAPTA‐AM could be used to prolong the sperm motility during cooling without apparently compromising fertility. Field trials should be conducted to assess fertility of cryopreserved stallion semen with BAPTA‐AM.     

Epigallocatechin‐3‐gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24–72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin‐3‐gallate (EGCG) at 20, 60 and 120 μm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

Acupuncture prevents the postpartum reduction in matrix metalloproteinase type‐2 immunoexpression,tissue concentration and enzyme activity in bovine caruncles

The objectives of this study were to determine the effects of acupuncture in dairy cows (Bos taurus) on caruncular matrix metalloproteinase type‐2 (MMP2) at 0, 2 and 4 hr after calving. Acupuncture (n = 6) was applied at 0 and 2 hr after calving to 6 points that relax the cervix and stimulate uterine contractions. Controls (n = 9) were kept in a stanchion for 15 min without acupuncture. All of the cows in the study delivered their placenta in <4 hr. Formalin‐fixed caruncles were paraffin‐embedded and subjected to routine immunohistochemistry to determine MMP2 expression, which was scored by a single observer. Flash frozen caruncles were homogenized, and protein concentration was determined. MMP2 concentrations were calculated using commercial bovine ELISAs. MMP2 enzyme activity was determined using zymography. The mean value for each time point for each cow was used to calculate the mean ± SEM for each treatment group. MMP2 was predominantly localized to the epithelial and subepithelial stromal cells of the caruncles in both treatment groups. MMP2 immunoexpression was lower 4 hr after calving in the control cows (p = 0.012) but not in the acupuncture treated cows indicating that acupuncture treatment maintained MMP expression. MMP2 tissue concentration was lower 2 hr after calving in the control cows (p = 0.048) but not in the acupuncture treated cows. MMP2 enzyme activity decreased from 0 to 2 hr after calving in control cows (p = 0.046) but not in acupuncture treated cows. This study provides physiologic evidence for the effects of acupuncture on the bovine reproductive tract and substantiates the use of this treatment in cases of placental retention.     

Influence of Bacillus subtilis GCB‐13‐001 on growth performance,nutrient digestibility,blood characteristics,faecal microbiota and faecal score in weanling pigs

The present study investigated the influence of Bacillus subtilis GCB‐13‐001 on growth performance, nutrient digestibility, blood characteristics, faecal microbiota and faecal score in weanling pigs. A total of 120 weaning pigs [(Landrace × Yorkshire) × Duroc; 7.73 ± 0.75 kg (28 days of age)] were randomly allotted into three treatments according to their initial body weight (BW) and gender in a 6‐week experiment. There were 8 replication pens in each treatment, with five pigs/pen. Dietary treatment groups were as follows: (a) basal diet (CON), (b) CON + 0.1% Bacillus subtilis GCB‐13‐001 1 × 10⁸ CFU/kg (T1) and (c) CON + 0.1% Bacillus subtilis GCB‐13‐001 1 × 10⁹ CFU/kg (T2). Days 1 to 7, the BW and ADG with T2 treatment were higher (p < .05) than CON treatment, as well as F:G showed trends in linear reduction (p < .1). Days 8 to 21, the BW and ADG were improved (p < .05) in pigs offered T1 and T2 diets compared with CON diet. Days 22 to 42, BW and ADG were higher (p < .05) in pigs fed T2 diet than CON and T1 diets, and the pigs fed T1 diet had higher BW than CON treatment. Overall, the ADG with the T2 treatment was higher (p < .05) than that with the T1 and CON treatments, and pigs offered T1 treatment had higher (p < .05) ADG than CON treatment. Moreover, F:G ratio were significantly decreased (p < .05) by T2 treatment compared with CON treatment. The faecal Lactobacillus counts were improved, and E. coli counts were reduced (p < .05) in pigs fed T2 diet compared with CON at the end of the experiment. In conclusion, supplementation of 0.1% Bacillus subtilis GCB‐13‐001 1 × 10⁹ CFU/kg has shown a beneficial effect in improving BW, increase ADG, decrease F:G ratio.