Changes in c‐erbB‐2 Immunoexpression in Feline Endometrial Adenocarcinomas

Human epidermal growth factor receptor type 2 (c‐erbB‐2), an oncoprotein with potential prognostic marker and therapeutic use, is overexpressed in several human and animal tumours. But information regarding this molecule in feline tumours is scarce. This study aimed to assess the changes in the immunohistochemical expression of c‐erbB‐2 in feline endometrial adenocarcinomas (FEA) compared to normal endometrium. An immunohistochemistry assay using a specific antibody against c‐erbB‐2 was performed in FEA samples (n = 34) and in normal endometrium in the follicular (FS; n = 12) and luteal (LS; n = 11) stages. In FEA, the c‐erbB‐2 immunoexpression was assessed in neoplastic epithelial cells whilst in normal endometria it was individually evaluated in the surface and the superficial and deep glandular epithelia (SE, SGE and DGE, respectively). In FS and in LS, all the epithelia were positive for c‐erbB‐2; positivity was higher in the SE and the SGE than in DGE. Twenty of the 34 FEA samples (58.8%) were positive for c‐erbB‐2 immunolabelling. Nevertheless, its expression was higher in all the epithelia in the FS compared to FEA (p ≤ 0.0001) or the LS (p = 0.016). The results presented herein suggest that c‐erbB‐2 molecule is differently expressed in the feline endometrium through the oestrous cycle and though it may also be involved in feline endometrial carcinogenesis, a question remains unanswered on the importance of additional pathways of epithelial proliferation in the neoplastic changes in feline endometrium.     

Expression of Heparin‐Binding EGF‐Like Growth Factor (HB‐EGF) in Bovine Endometrium: Effects of HB‐EGF and Interferon‐τ on Prostaglandin Production

Heparin‐binding EGF‐like growth factor (HB‐EGF) regulates several cell functions by binding to its membrane receptor (ErbB1 and ErbB4). Experimental evidences suggest that HB‐EGF, prostaglandins (PGs) and interferon‐τ (IFN‐τ) regulate uterine function for pregnancy establishment in ruminants. In this study, the mRNA expressions of HB‐EGF, ErbB1 and ErbB4 in bovine endometrium and the effects of HB‐EGF and IFN‐τ on PGE2 and PGF2‐α production by endometrial cells were investigated. RT‐PCR analysis revealed that HB‐EGF mRNA was greater at the mid‐luteal stage than at the early and regressed luteal stages (p < 0.05). ErbB1 mRNA expression was greater at the mid‐ and late luteal stages than at the other luteal stages (p < 0.05). IFN‐τ increased the expression of HB‐EGF, ErbB1 and ErbB4 mRNA in epithelial cells (p < 0.05). HB‐EGF did not affect PGF2‐α or PGE2 production by bovine endometrial epithelial cells, but increased PGF2‐α and PGE2 production by bovine endometrial stromal cells (p < 0.05). IFN‐τ significantly decreased HB‐EGF‐stimulated PGF2‐α (p < 0.05), but not PGE2 (p > 0.05) production by stromal cells. These results indicate that HB‐EGF and its receptors expression changed in bovine endometrium throughout the oestrous cycle. IFN‐τ increased their expression in cultured endometrial cells. HB‐EGF and IFN‐τ have the ability to regulate PGs production by stromal cells and therefore may play a role in the local regulation of uterine function at the time of implantation in cattle.     

Peri‐conceptional under‐nutrition alters the expression of TRIM28 and ZFP57 in the endometrium and embryos during peri‐implantation period in domestic pigs

DNA methylation is maintained by the main elements of methylation complex—tripartite motif containing 28 (TRIM28) and zinc finger protein 57 (ZFP57). Previously, it was found that the activity of TRIM28 and ZFP57 determines the process of DNA methylation and preserves over‐expression of genes. We hypothesized that restricted diet applied during peri‐conceptional period may induce changes in the expression of methylation complex in porcine endometrium and embryos during the peri‐implantation period. The aim of this study was to detect and determine the expression of TRIM28 and ZFP57 in the endometrium and embryos harvested from gilts during the peri‐implantation period (days 15–16 of pregnancy) fed restricted (n = 5) or normal (n = 5) diet during peri‐conceptional period. In restricted‐diet‐fed gilts, endometrial expression of TRIM28 and ZFP57 mRNAs was decreased in comparison with normal‐diet‐fed gilts (p ≤ .01), while the embryonic expression of TRIM28 and ZFP57 mRNAs was increased in restricted‐diet‐fed gilts (p ≤ .05). The immunofluorescence showed the presence of TRIM28 and ZFP57 in luminal epithelial (LE), glandular epithelial (GE) and stromal cells (ST) of the endometrium as well as in the embryos. Total endometrial and embryonic abundance of TRIM28 and ZFP57 proteins was significantly higher (p ≤ .05) in restricted‐diet‐fed gilts than in normal‐diet‐fed gilts. Female under‐nutrition during peri‐conceptional period affects the expression of two main elements of methylation complex in the endometrium and in embryos during the peri‐implantation period and may have the impact on DNA methylation in these tissues.     

Oestrogen and Progesterone Receptors and COX‐2 Expression in Endometrial Biopsy Samples During Maternal Recognition of Pregnancy in Llamas (Lama glama)

Endometrial expression of oestrogen receptor‐α (ERα), progesterone receptor (PR) and cyclooxigenase‐2 (COX‐2) was evaluated in non‐pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non‐pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post‐induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post‐mating and by progesterone profile on day 12 post‐mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX‐2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non‐pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post‐induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX‐2 expression was lower in pregnant than in non‐pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF_2α secretion during early pregnancy by decreasing the endometrial expression of COX‐2 in the luminal epithelium of pregnant llamas.     

Effects of Interferon‐Tau and Steroids on Cytochrome P450 Activity in Bovine Endometrial Epithelial Cells

The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon‐τ (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were treated with OT, IFN, a combination of OT+IFN or control (CON) media for 24 h. For the second experiment, cells were treated with E2, P4, a combination of E2 + P4 or CON media for 24 h. Treatments were performed in triplicate, and the experiment was repeated four times (n = 12 per treatment). Treatment with OT alone increased (p < 0.01) activity of COX compared with CON; however, OT alone did not alter activity of CYP1A (p = 0.55) or CYP2C (p = 0.46) compared with CON. Activity of CYP1A and CYP2C was decreased in cells exposed to IFN (p < 0.01) or OT+IFN (p < 0.01) compared with CON. Treatment with E2 alone did not alter activity of CYP1A (p = 0.64) or CYP2C (p = 0.06) compared with CON. Activity of CYP1A and CYP2C was decreased (p < 0.01) in P4 vs CON. In summary, IFN exposure, irrespective of OT treatment, decreased the activity of CYP1A and CYP2C. Activity of CYP1A was decreased following P4 treatment alone, while that of CYP2C was decreased following both P4 and E2 + P4 treatment. The mixed function monooxygenase enzymes, CYP1A and CYP2C, have been implicated in synthesizing embryotoxic compounds; therefore, downregulation in the endometrium may be necessary during maternal recognition of pregnancy.     

Investigations on the Endometrial Response to Intrauterine Administration of N‐Acetylcysteine in Oestrous Mares

In mares, mating‐induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N‐acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki‐67), lectins and periodic acid‐Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC‐ and C‐mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC‐treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki‐67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC‐treated mares than in C‐mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti‐inflammatory effect on the equine endometrium was observed.     

Measurement of C‐reactive protein and Prostaglandin F2α Metabolite Concentrations in Differentiation of Canine Pyometra and Cystic Endometrial Hyperplasia/Mucometra

Canine pyometra is a dioestrus period disease in which systemic inflammatory response syndrome (SIRS) is a common outcome due to the response of the body to the bacterial infection. The purpose of this study was i) to differentiate canine pyometra and cystic endometrial hyperplasia (CEH)/mucometra by measuring serum C‐reactive protein (CRP) and prostaglandin F_2α metabolite (PGFM) concentrations in blood and ii) to compare serum concentrations of CRP and PGFM in bitches with a pathological uterus (pyometra or CEH/mucometra) to concentrations in bitches with a healthy uterus. Mean CRP concentrations were found significantly higher (p < 0.001) in dogs with pyometra compared to those with CEH/mucometra or healthy uterus. However, no statistical difference could be detected between the groups for mean PGFM concentrations. Mean white blood cell count (WBC), alkaline phosphatase (ALP) and total protein concentrations were found significantly higher (p < 0.001) in dogs with pyometra. Escherichia coli was the most frequently isolated microorganism from dogs with pyometra (64.3%). Edwardsiella spp. was detected in a single case of pyometra for the first time. In conclusion, our results demonstrate that serum CRP concentrations were increased in dogs with pyometra and thus we conclude that serum CRP concentration but not PGFM might be useful as a marker to differentiate a case of CEH/mucometra from pyometra in female dogs. To the authors' knowledge, this is the first report in which Edwardsiella spp. has been isolated in the canine uterus.     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

Investigation of the usability of kisspeptin and oxidative stress parameters in the early diagnosis of asymptomatic cystic endometrial hyperplasia in dogs

This study aimed to investigate the differences in oxidative stress index (OSI) and kisspeptin levels in clinically asymptomatic dogs with cystic endometrial hyperplasia (CEH) compared to healthy and pregnant dogs, and to determine the usability of the obtained results in the diagnosis of asymptomatic CEH. The study comprised three groups; a healthy (n = 8), a pregnant (n = 10) and a CEH (n = 10). All dogs in the three groups were included in the study at the 30 ± 3th day after estrus, and blood samples were collected for analysis of kisspeptin, total antioxidant status (TAS), total oxidant status (TOS), progesterone (P4), estradiol (E2) and some biochemical parameters (TSH; thyroid stimulating hormone, ALT; alanine aminotransferase, AST; aspartate aminotransferase, ALP; alkaline phosphatase, LDH; lactate dehydrogenase, CRE; creatine and BUN; blood urea nitrogen). In addition, OSI value was calculated. P4 and ALT and BUN levels were significantly lower and higher in CEH group than the pregnant group, respectively (p < .05). While kisspeptin and TAS levels were significantly lower in CEH group compared to the healthy and pregnant groups (p < .01), OSI level increased dramatically. In conclusion, it was confirmed that CEH clearly affected kisspeptin and OSI levels, and it is thought that these parameters may be an alternative diagnostic tool for the detection of CEH after further studies.     

IFN‐τ Acts in a Dose‐Dependent Manner on Prostaglandin Production by Buffalo Endometrial Stromal Cells Cultured in vitro

Interferon‐τ (IFN‐τ) has been recognized as the primary embryonic signal responsible for maternal recognition of pregnancy. Uterine endometrium produces both prostaglandin F_2α (PGF_2α) and prostaglandin E₂ (PGE₂). PGF_2α is responsible for the luteolysis; however, PGE₂ favours establishment of pregnancy by its luteoprotective action. In this study, the dose‐response effect of recombinant bovine IFN‐τ (rbIFN‐τ) on prostaglandin (PG) production by buffalo endometrial stromal cells cultured in vitro was studied. Buffalo endometrial stromal cells were isolated by double enzymatic digestion, initially with trypsin III followed by a cocktail of trypsin III, collagenase type II and DNase I and subsequently cultured till confluence. Further, cells were treated with different doses of rbIFN‐τ (0.001, 0.01, 0.1, 1.0 and 10 μg/ml) and keeping a separate set of control. Culture supernatant was collected after 6, 12 and 24 h of treatment. PG levels in the culture supernatant were measured by enzyme immune assay (EIA) and total cellular protein estimated by Bradford method. Results indicated that buffalo endometrial stromal cells following rbIFN‐τ treatment enhanced the secretion of both PGE₂ and PGF_2α, and also its ratio in a strict dose‐dependent manner with a significant increase (p < 0.01) in PGE₂ production at 1 μg/ml dose of rbIFN‐τ and maximal stimulation for both PG was observed at 10 μg/ml. Further, both PG production and its ratio were increased significantly (p < 0.01) in a time‐dependent fashion in all the groups at 6, 12 and 24 h post‐treatment with highest level achieved at 24 h as compared with control. Absolute levels of PGE₂ remained higher than PGF_2α indicating PGE₂ as the major PG produced by endometrial stromal cells. The dose‐dependent response of rbIFN‐τ signifies the importance of optimum concentration of IFN‐τ for the embryonic development especially during the critical period to establish successful pregnancy.     

Expression and regulation of cyclooxygenase-2 in normal and neoplastic canine keratinocytes

Squamous cell carcinoma (SCC) is one of the most common cancers in dogs, yet relatively little is known about the molecular events involved in its development. Increasing evidence implicates cyclooxygenase‐2 (COX‐2) in the pathogenesis of various cancers in humans and animals. COX‐2 overexpression has recently been demonstrated in canine SCCs. The objective of our study was to characterize the expression and regulation of COX‐2 in normal and neoplastic canine keratinocytes (CKs) to provide an in vitro system to investigate the implication of COX‐2 in SCC oncogenesis in dogs. Cell lines derived from normal CKs and neoplastic CKs (SCCs) were cultured in the absence or presence of agonists, and immunoblots, immunocytochemistry, radioimmunoassays and a cell proliferation assay were used to characterize COX‐2 expression and action. Results showed that neoplastic keratinocytes had a higher basal COX‐2 expression than normal keratinocytes. In both cell lines, stimulation with the tumour promoter phorbol 12‐myristate 13‐acetate induced a time‐dependent increase in COX‐2 protein, with COX‐2 induction being stronger in cancerous SCC than in normal CK cells. Moreover, SCC cells produced significantly more PGE₂ than CK cells, under both baseline and stimulated conditions (P < 0.05). NS‐398, a selective COX‐2 inhibitor, inhibited prostaglandin (PG)E₂ synthesis and decreased proliferation of CK and SCC cells (P < 0.05). Collectively, our results indicate that the canine neoplastic keratinocyte SCC cell line expresses more COX‐2 and produces more PGE₂ than the normal keratinocyte CK cell line, thus providing an in vitro system to study the molecular basis of elevated COX‐2 expression in SCCs in dogs.     

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.     

Retrospective evaluation of COX‐2 expression,histological and clinical factors as prognostic indicators in dogs with renal cell carcinomas undergoing nephrectomy

Limited veterinary literature is available regarding prognostic markers for canine renal cell carcinoma (CRCC). We retrospectively evaluated COX‐2 expression, histological and clinical features associated with prognosis of CRCC. Sixty‐four cases post‐nephrectomy were included, 54 had histopathological assessment and 30 had COX‐2 immunostaining performed. Eight dogs (13%) had metastatic disease at initial diagnosis. Twenty‐seven dogs (42%) received adjuvant therapy after nephrectomy. On univariate analysis, COX‐2 expression, mitotic index (MI), histologic type, vascular invasion, neoplastic invasiveness and metastasis at diagnosis were significantly associated with overall median survival time (MST). COX‐2 score (COX‐2 score > 3 MST 420 days versus 1176 days if COX‐2 score <3; P = 0.011) and MI (MI > 30 MST 120 days versus 540 days for MI < 30; P = 0.003) were the only variables associated with CRCC outcome on multivariate analysis. The addition of MI and COX‐2 immunostaining to standard histopathological evaluation would help predicting outcome in CRCC patients.     

Enhanced expression of cyclooxygenase-2 in glaucomatous dog eyes

Objective Cyclooxygenase‐2 (COX‐2)‐derived prostaglandins (PGs) are shown to play important pathophysiologic roles in various disease states. Recently, the effectiveness of topical PGs in reducing intraocular pressure (IOP) has stimulated further interest in the physiologic function of COX‐2 and PGs in normal and glaucomatous eyes. Therefore, we investigated the cell‐type distribution and expression of COX‐2 in normal and glaucomatous dog eyes. Procedures Using isoform‐specific antibodies, we immunohistochemically evaluated COX‐2 expression in formalin‐fixed and paraffin‐embedded normal (n = 5) and glaucomatous (n = 17) dog eyes. Results In the normal eyes, only minimal COX‐2 immunoreactivity was observed in the ciliary epithelium. In the glaucomatous eyes, COX‐2 expression was further observed in the cornea and corneoscleral limbus. In the cornea, moderate to strong COX‐2 expression was observed in all corneal layers (epithelium, stromal cells and endothelium), with the greatest expression present in the epithelial layer. In the corneoscleral limbus area, COX‐2 immunoreactivity was noted in the stromal cells of sclera, trabecular meshwork and endothelial cells of the angular aqueous plexus. Conclusions Increased expression of COX‐2 in dog glaucomatous eyes suggests that COX‐2‐derived PGs may have a potential role in the pathogenesis of canine glaucoma.     

In vitro enantioselective pharmacodynamics of Carprofen and Flunixin‐meglumine in feedlot cattle

The activity of the anti‐inflammatory agents Flunixin‐meglumine (FLU), RS (±) Carprofen (CPF) and S (+) CPF on bovine cyclooxygenases (COXs) has been characterized in feedlot calves using an in vitro whole blood model. The drugs showed equivalent efficacy in their inhibitory activity on COXs, and the rank order of potency for both COX‐1 and COX‐2 inhibition was FLU > S (+) CPF > RS (±) CPF. Our results indicated that FLU is a nonselective inhibitor of bovine COXs, whereas RS (±) CPF and S (+) CPF exhibited different degrees of preferential inhibition of COX‐2 isoenzyme. The rank order of IC₅₀ COX‐1: IC₅₀ COX‐2 potency ratios was in fact S (+) CPF (51.882) > RS (±) CPF (13.964) > FLU (0.606), and the calculated percentage inhibition of COX‐1 corresponding to COX‐2 inhibition values comprised between 80% and 95% was comprised between 57.697 and 79.865 for FLU, 33.373 and 51.319 for RS (±) CPF, and 0.230 and 4.622 for S (+) CPF, respectively. These findings are discussed in relation to the prediction of the clinical relevance of COX inhibition by the test drugs in cattle.     

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.     

Molecular cloning and expression of FGF2 gene in pre‐implantation developmental stages of in vitro‐produced sheep embryos

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor‐2 (FGF‐2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non‐pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO₂, 95% humidity for 22–24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO₂ incubator for 6–7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real‐time polymerase chain reaction using sequence‐specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2‐cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre‐implantation stages of embryo. It can be concluded that FGF‐2 plays a significant role in pre‐implantation and early development of embryos in sheep.     

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.