Neoplastic transformation of porcine mammary epithelial cells in vitro and tumor formation in vivo.

BackgroundThe mammary glands of pigs share many functional and morphological similarities with the breasts of humans, raising the potential of their utility for research into the mechanisms underlying normal mammary function and breast carcinogenesis. Here we sought to establish a model for the efficient manipulation and transformation of porcine mammary epithelial cells (pMEC) in vitro and tumor growth in vivo.MethodsWe utilized a vector encoding the red florescent protein tdTomato to transduce populations of pMEC from Yorkshire -Hampshire crossbred female pigs in vitro and in vivo. Populations of primary pMEC were then separated by FACS using markers to distinguish epithelial cells (CD140a-) from stromal cells (CD140a+), with or without further enrichment for basal and luminal progenitor cells (CD49f+). These separated pMEC populations were transduced by lentivirus encoding murine polyomavirus T antigens (Tag) and tdTomato and engrafted to orthotopic or ectopic sites in immunodeficient NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ (NSG) mice.ResultsWe demonstrated that lentivirus effectively transduces pMEC in vitro and in vivo. We further established that lentivirus can be used for oncogenic-transformation of pMEC ex vivo for generating mammary tumors in vivo. Oncogenic transformation was confirmed in vitro by anchorage-independent growth, increased cell proliferation, and expression of CDKN2A, cyclin A2 and p53 alongside decreased phosphorylation of Rb. Moreover, Tag-transformed CD140a- and CD140a-CD49f + pMECs developed site-specific tumors of differing histopathologies in vivo.ConclusionsHerein we establish a model for the transduction and oncogenic transformation of pMEC. This is the first report describing a porcine model of mammary epithelial cell tumorigenesis that can be applied to the study of human breast cancers

Effect of site of sperm deposition on fertility when sows are inseminated with aged semen

Abstract in English, Spanish and FrenchWith conventional insemination, farrowing rate and litter size were lower (P < .05) when sperm was aged (≥ 4 days; n = 30) rather than fresh (≤ 3 days; n = 29). Farrowing rate, but not litter size, was maintained with intrauterine insemination of aged sperm (n = 29). = Con las inseminación convencional, el porcentaje de fertilidad y el tamaño de la camada fueron menores (P < .05) cuando el esperma era viejo (≥ 4 días; n = 30) en vez de fresco (≤ 3 días; n = 29). El porcentaje de fertildiad, pero no el tamaño de la camada, se mantuvo con la inseminación intrauterina de semen viejo (n = 29). = Lors d’insémination conventionnelle, le taux de mise-bas et la taille des portées étaient plus faibles (P < .05) lorsque le sperme était âgé (≥ 4 jours; n = 30) plutôt que frais (≤ 3 jours; n = 29). Le taux de mise-bas, mais pas la taille des portées, était maintenu avec l’insémination intra-utérine de sperme âgé (n = 29).Nutthee Am-in, Wichai Tantasuparuk, R. Manjarin, R. N. Kirkwoo

Effect of thawing frozen boar semen in 50% seminal plasma on sow fertility

To examine the effects of seminal plasma, 97 multiparous Yorkshire sows each received 900 IUeCG at weaning and then 5mg pLH 80 hours later to control time of ovulation.At 36 hours after pLH, sows were assigned on the basis of parity to receive a single intrauterine insemination of 3 x 109 fresh sperm(CON) or 5 x 109 cryo-preserved sperm thawed (FT) into 80mL extender without FT or with 50% Seminal Plasma (SP) (FT-SP).Compared with CONsows, FT-SP sows had decreased (P<0.01) farrowing rate and FT sows were intermediate. Litter sizes were unaffected by treatment.J.C. Garcia, J. C. Dominguez, R. Manjarin, B. Alegre, R.N. Kirkwoo

Transcript abundance of hormone receptors, mammalian target of rapamycin pathway-related kinases, insulin-like growth factor I, and milk proteins in porcine mammary tissue

Prolactin, glucocorticoids, and insulin are commonly used to induce milk protein synthesis in bovine mammary cell cultures. In addition, administration of GH increases milk yield in dairy cows, likely via the mammalian target of rapamycin (mTOR) pathway and IGF-I synthesis. As such, the hypothesis of this study was that mRNA abundance of hormone receptors, mammalian target of mTOR pathway-related kinases, IGF-I, and milk protein-encoding genes increases in the porcine mammary gland in response to greater lactation demand. Selected genes included those encoding for receptors of GH (GHR), insulin (INSR), glucocorticoid (NR3C1), prolactin (PRLR), IGF-I (IGF-I), mTOR (FRAP1), and p70S6 kinases (RPS6KB1), and the milk proteins α-lactalbumin (LALBA) and β-casein (CSN2). Mammary tissue was biopsied from 4 sows on d 110 of gestation (prepartum), d 5 and 17 of lactation, and d 5 after weaning (postweaning), and gene expression was quantified by reverse-transcription quantitative PCR. Compared with prepartum, d 5 of lactation increased (P < 0.001) NR3C1, tended to increase (P = 0.06) GHR, and decreased (P < 0.001) PRLR mRNA abundance. Compared with d 5 of lactation, d 17 of lactation increased PRLR (P < 0.001) and decreased GHR (P < 0.01). Expression of INR and FRAP1 did not differ when comparing either prepartum or d 17 of lactation with d 5 of lactation. Compared with d 17 of lactation, postweaning decreased (P < 0.001) PRLR, did not affect INSR, and increased both IGF-I and GHR (P < 0.05) mRNA abundance. From prepartum to d 17 of lactation, NR3C1 mRNA abundance was positively correlated with CSN2 (r = 0.85; P < 0.001) and LALBA mRNA abundance (r = 0.79; P = 0.002), whereas mRNA abundance of GHR tended to be positively correlated with that of IGF-I (r = 0.46; P = 0.06). In conclusion, expression of the genes NR3C1, PRLR, GHR, and IGF-I changed in the porcine mammary gland during the prepartum to postweaning periods, but only NR3C1 mRNA abundance was positively correlated with expression of CSN2 and LALBA.R. Manjarín, J. P. Steibel, R. N. Kirkwood, N. P. Taylor, and N. L. Trottie

Effect of hCG treatment on the oestrous and ovulation responses to FSH in prepubertal gilts

To ensure sufficient numbers of pregnant females, particularly at hotter times of the year, hormonal induction of gilt oestrus may be necessary. However, the gilt oestrus and ovulation responses to gonadotrophin treatment have often proven unpredictable. The objective of this study was to examine possible reasons for this unpredictability. Prepubertal gilts (approximately 150 days of age, n = 63) were assigned to one of three treatments: injection of 300 IU hCG (n = 15); pre-treatment with 100 mg FSH in polyvinylpyrrolidinone administered as 2 × 50 mg injections 24 h apart, followed by 600 IU eCG at 24 h after the second FSH injection (n = 23); or FSH pre-treatment as above followed by 300 IU hCG at 24 h after the second FSH injection (n = 25). To facilitate oestrus detection, gilts were exposed to a mature boar for 15 min daily for 7 days. Blood samples were obtained on the day of eCG or hCG injection and again 10 days later and gilt ovulation responses determined based on elevated progesterone concentrations. The oestrus responses by 7 days were 6.7%, 17.5% and 64.0% for gilts treated with hCG, FSH + eCG and FSH + hCG, respectively (p < 0.001). The oestrous gilt receiving hCG alone and one oestrous FSH + hCG gilt did not ovulate, all other oestrous gilts ovulated. A further two anoestrous FSH + eCG-treated gilts ovulated. These data suggest that FSH pre-treatment facilitated the development of ovarian follicles to the point where they became responsive to hCG, but had little effect on the response to eCG.R Manjarin, JC Dominguez, MJ Castro, DJ Sprecher, G Cassar, RM Friendship and RN Kirkwoo

Effect of eCG or eCG plus hCG on oestrus expression and ovulation in prepubertal gilts

To meet weekly breeding targets, it is occasionally necessary to inject exogenous gonadotrophins to induce oestrus in prepubertal gilts. However, the gilt oestrus response to equine chorionic gonadotrophin (eCG) either alone or in combination with human chorionic gonadotrophin (hCG) can be unpredictable. The objective of the present study was to examine possible reasons for this unpredictability. Prepubertal gilts (90 kg and 153 days of age, n = 109) received an injection of either 600 IU eCG or a combination of 400 IU eCG and 200 IU hCG (PG600), or were non-injected controls, and were then exposed to a mature boar for 15 min daily for 7 days for oestrus detection. At the time of injection, real-time ultrasound revealed that the gilt ovaries had primarily 1–2 mm follicles. Blood samples were obtained at time of hormone injection (day 0) and at days 3, 7 and 10 for assay of serum progesterone concentrations. The oestrus responses by 7 days were15.5%, 73.3% and 0%, for eCG, PG600, and control gilts, respectively (p < 0.001). The oestrus response improved (p < 0.05) with increasing body weight. Based on circulating progesterone levels, all oestrous gilts ovulated except for four of the PG600 gilts. Failure to express oestrus in PG600 gilts was not associated with a premature rise in progesterone.R Manjarin, G Cassar, DJ Sprecher, RM Friendship, JC Dominguez and RN Kirkwoo

Relationship between vaginal mucus conductivity and time of ovulation in weaned sows

This study investigated whether changes in the vaginal electrical resistance (VER) of vaginal mucus of weaned sows during the first 7 d post-weaning are associated with time of ovulation. Time of ovulation was determined by ovarian ultrasound carried out from 91 to 146 h after weaning and at different seasons. Vaginal electrical resistance was measured at 20, 44, 68, 91, 96, 102, 115, 120, 126, 140, 146, and 164 h post-weaning and was found to decrease between 120 h and 31 h before ovulation and then increase until 40 to 50 h after ovulation. Duration and timing of the nadir was affected by the season (P < 0.01). Estrus was observed from day 4 after the lowest VER values. Ovulation occurred between late day 5 and late day 6, while VER values were still increasing. Ovulation was earlier in lower parity sows (P < 0.001). Compared to 0 h (ovulation time), VER was significantly lower from 50 to 5 h before ovulation in autumn and from 40 to 21 h in winter, but such differences were not seen in spring. Lowest VER value was not correlated with time of ovulation. It was concluded that VER increases before ovulation and, although this increase is influenced by the season, it cannot be used to accurately predict ovulation in weaned sows.David M. Hidalgo, Glen Cassar, Rodrigo Manjarin, Juan C. Dominguez, Robert M. Friendship, Roy N. Kirkwoo

Influence of lactation length and gonadotrophins administered at weaning on fertility of primiparous sows

Abstract not availableD.M. Hidalgo, R.M. Friendship, L. Greiner, R. Manjarin, M.R. Amezcua, J.C. Dominguez, R.N. Kirkwoo

%polynova_2way: A SAS macro for implementation of mixed models for metabolomics data.

The generation of large metabolomic data sets has created a high demand for software that can fit statistical models to one-metabolite-at-a-time on hundreds of metabolites. We provide the %polynova_2way macro in SAS to identify metabolites differentially expressed in study designs with a two-way factorial treatment and hierarchical design structure. For each metabolite, the macro calculates the least squares means using a linear mixed model with fixed and random effects, runs a 2-way ANOVA, corrects the P-values for the number of metabolites using the false discovery rate or Bonferroni procedure, and calculate the P-value for the least squares mean differences for each metabolite. Finally, the %polynova_2way macro outputs a table in excel format that combines all the results to facilitate the identification of significant metabolites for each factor. The macro code is freely available in the Supporting Information