PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Moura C.E.B., Albuquerque J.F.G., Magalhães M.S., Silva N.B., Oliveira M.F. & Papa P.C. 2007. [Comparative analysis of the origin of the brachial plexus of the collared peccary (Tayassu tajacu).] Análise comparativa da origem do plexo braquial de catetos (Tayassu tajacu). Pesquisa Veterinária Brasileira 27(9):357-362. Departamento de Morfologia, Universidade Federal do Rio Grande do Norte, Cx. Postal 1524, Campus Universitário Lagoa Nova, Natal, RN 59072-970, Brazil. E-mail: cadumoura@ufrnet.br Collared peccary (Tayassu tajacu) belongs to the Tayassuidae family, characterized by a “collar” of white hairs that cross behind the neck and extend bilaterally in front of the shoulders. It can be found from south-western United States to Argentina. In the literature a shortage of data is verified regarding the functional anatomy of the collared peccaries, especially of studies that involve the anatomy of the brachial plexus. To elucidate the behavior of this plexus of collared peccaries and with the purpose to contribute for the development of compared anatomy, this study was accomplished. Thirty animals of different ages were used (17 males and 13 females) coming from the Wild Animal Multiplication Center of the “Universidade Federal Rural do Semi-árido” Mossoró, Rio Grande do Norte, Brazil. After slaughter bilateral dissection of the brachial plexuses took place, and the results were registered in schematic drawings and the dispositions grouped in tables for subsequent statistical analysis based on the percentile frequency. It was found that the Plexus brachialis of collared peccaries is the result of established communications, mainly among the Rami ventrales of the last three cervical nerves and of the first two thoracic nerves, having a contribution of the fourth and fifth cervical nerves in 16.67% and 50.00% of the cases, respectively. In 40.00% of the dissections the most frequent plexus was of the type C6, C7, C8, T1 and T2. The main nerves derived from brachial plexus of the collared peccaries and its respective origins had been: Nervus suprascapularis (C6, C7), Nn. subscapulares (C5, C6 e C7 or C6 e C7), N. axillaris (C6, C7), N. musculocutaneus (C7, C8), N. medianus (C7, C8, T1, T2), N. radialis (C8, T1, T2), N. ulnaris (C8, T1, T2), cranialis (C7), and caudalis (C7, C8) Nn. pectorales, N. thoracodorsalis (C6, C7, C8), N. thoracicus longus (C7, C8), and N. thoracicus lateralis (C8, T1, T2).

• Brachial plexus neuroanatomy wild animals

Abstract in Portuguese:

ABSTRACT.- Moura C.E.B., Albuquerque J.F.G., Magalhães M.S., Silva N.B., Oliveira M.F. & Papa P.C. 2007. [Comparative analysis of the origin of the brachial plexus of the collared peccary (Tayassu tajacu).] Análise comparativa da origem do plexo braquial de catetos (Tayassu tajacu). Pesquisa Veterinária Brasileira 27(9):357-362. Departamento de Morfologia, Universidade Federal do Rio Grande do Norte, Cx. Postal 1524, Campus Universitário Lagoa Nova, Natal, RN 59072-970, Brazil. E-mail: cadumoura@ufrnet.br Collared peccary (Tayassu tajacu) belongs to the Tayassuidae family, characterized by a “collar” of white hairs that cross behind the neck and extend bilaterally in front of the shoulders. It can be found from south-western United States to Argentina. In the literature a shortage of data is verified regarding the functional anatomy of the collared peccaries, especially of studies that involve the anatomy of the brachial plexus. To elucidate the behavior of this plexus of collared peccaries and with the purpose to contribute for the development of compared anatomy, this study was accomplished. Thirty animals of different ages were used (17 males and 13 females) coming from the Wild Animal Multiplication Center of the “Universidade Federal Rural do Semi-árido” Mossoró, Rio Grande do Norte, Brazil. After slaughter bilateral dissection of the brachial plexuses took place, and the results were registered in schematic drawings and the dispositions grouped in tables for subsequent statistical analysis based on the percentile frequency. It was found that the Plexus brachialis of collared peccaries is the result of established communications, mainly among the Rami ventrales of the last three cervical nerves and of the first two thoracic nerves, having a contribution of the fourth and fifth cervical nerves in 16.67% and 50.00% of the cases, respectively. In 40.00% of the dissections the most frequent plexus was of the type C6, C7, C8, T1 and T2. The main nerves derived from brachial plexus of the collared peccaries and its respective origins had been: Nervus suprascapularis (C6, C7), Nn. subscapulares (C5, C6 e C7 or C6 e C7), N. axillaris (C6, C7), N. musculocutaneus (C7, C8), N. medianus (C7, C8, T1, T2), N. radialis (C8, T1, T2), N. ulnaris (C8, T1, T2), cranialis (C7), and caudalis (C7, C8) Nn. pectorales, N. thoracodorsalis (C6, C7, C8), N. thoracicus longus (C7, C8), and N. thoracicus lateralis (C8, T1, T2).

Abstract in English:

ABSTRACT.- Faleiros R.R., Macoris D.G., Alves G.E.S., Saquetti C.H.C. & Alessi A.C. 2007. [Histomorphometric and ultrastructural evaluation of the mucosa of the equine small colon subjected to distention.] Avaliação histomorfométrica e ultra-estrutural da mucosa do cólon menor eqüino submetido à distensão. Pesquisa Veterinária Brasileira 27(9):383-387. Departamento de Clínica e Cirurgia Veterinária, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Campus de Jaboticabal, Rodovia Carlos Tonanni Km 5, Jaboticabal, SP 14870-000, Brazil. E-mail: faleiros@ufmg.br Recently it has been shown that experimental distention of the small colon of horses promotes reduction of microvascular circulation and inflammation of the seromuscular layer associated with neutrophil accumulation in the lungs. However this model was not sufficient to induce evident histophatological changes in the mucosal layer. The aim of this study was to evaluate the mucosa subjected to that model of small colon distention by histomorphometry and scan electronic microscopy (SEM). Sixteen horses were used. In the distended group (DG), nine of them were subjected to distention of the small colon by a surgically implanted intraluminal balloon that was inflated with a pressure of 40mm Hg during 4 hours. In the sham-operated group (SG), the balloon was implanted but not inflated. Full-thickness intestinal samples were collected before and after obstruction and after 1.5 and 12 hours of decom-pression. By SEM, it was observed that the mucosa turned flat and smooth after distention and returned to the wrinkled original appearance after decompression. Twelve hours after decompression the mucosa had a more irregular appearance with points of fragmentation. There was a reduction in mucosa thickness after distention, returning to basal values after decompression. Instead of the fact that there were changes in appearance and thickness, it was concluded that the mucosa could borne up the compression caused by distention returning to the original characteristics without major lesions.

• Small colon intestinal distention colic horse

Abstract in Portuguese:

ABSTRACT.- Faleiros R.R., Macoris D.G., Alves G.E.S., Saquetti C.H.C. & Alessi A.C. 2007. [Histomorphometric and ultrastructural evaluation of the mucosa of the equine small colon subjected to distention.] Avaliação histomorfométrica e ultra-estrutural da mucosa do cólon menor eqüino submetido à distensão. Pesquisa Veterinária Brasileira 27(9):383-387. Departamento de Clínica e Cirurgia Veterinária, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, Campus de Jaboticabal, Rodovia Carlos Tonanni Km 5, Jaboticabal, SP 14870-000, Brazil. E-mail: faleiros@ufmg.br Recently it has been shown that experimental distention of the small colon of horses promotes reduction of microvascular circulation and inflammation of the seromuscular layer associated with neutrophil accumulation in the lungs. However this model was not sufficient to induce evident histophatological changes in the mucosal layer. The aim of this study was to evaluate the mucosa subjected to that model of small colon distention by histomorphometry and scan electronic microscopy (SEM). Sixteen horses were used. In the distended group (DG), nine of them were subjected to distention of the small colon by a surgically implanted intraluminal balloon that was inflated with a pressure of 40mm Hg during 4 hours. In the sham-operated group (SG), the balloon was implanted but not inflated. Full-thickness intestinal samples were collected before and after obstruction and after 1.5 and 12 hours of decom-pression. By SEM, it was observed that the mucosa turned flat and smooth after distention and returned to the wrinkled original appearance after decompression. Twelve hours after decompression the mucosa had a more irregular appearance with points of fragmentation. There was a reduction in mucosa thickness after distention, returning to basal values after decompression. Instead of the fact that there were changes in appearance and thickness, it was concluded that the mucosa could borne up the compression caused by distention returning to the original characteristics without major lesions.

Abstract in English:

ABSTRACT.- Alves F.R., Costa F.B., Arouche M.M.S., Barros A.C.E., Miglino M.A., Vulcano L.C. & Guerra P.C. 2007. [Ultrasonographic evaluation of the urinary system, liver and uterus of Cebus apella monkey.] Avaliação ultra-sonográfica do sistema urinário, fígado e útero do macaco-prego, Cebus apella. Pesquisa Veterinária Brasileira 27(9):377-382. Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP 05508-270, Brazil. E-mail: flaviovet@usp.br The Brown Capuchin, Cebus apella, has a wide distribution in the northern and southern Brazilian Amazon region and in the Cerrado (savanna). These monkeys are usually submitted to predatory chase, increasing the need for preservation of this wild animal species. An ultrasonographic examination of 10 Brown Capuchins was made in order to describe the normal ultrasonographic anatomy of their abdominal cavity. The urinary bladder revealed its wall thickness with an average of 0.2cm, the topographic situation of which allowed close relation with the wall of uterus and descendent colon. Using caudal abdominal scan, images of aorta, caudal vena cava and right iliac vein were obtained. Liver was accessible for examination by sagittal and cross-section ultrasound, allowing visualization of gallbladder and hepatic vessels. Renal scan allowed accuracy to evidence the echogenicity differences between pelvis, renal sinus, as well as the cortical-medullary relationship. The mean length of the kidneys was 6.24±0.31cm, and no significant differences were observed between left and right kidney length (Student’s t-test and ANOVA). The renal volume obtained was 2.37±0.18cm3. Correlation Coefficients of Pearson between right and left renal length and between right and left renal volume were r = 0.74 and 0.51. Mean thickness for cortical and medullar regions was 0.75±0.11 and 0.39±0.06cm, respectively. Correlation Coefficient of corticomedullar relation between right and left renal was r = 0.19. Examination by ultrasound was considered an efficient, non-invasive, fast and repeatable technique which provides useful data for clinicians and surgeons engaged in wild animal medicine.

• Cebus apella Brown Capuchin ultrasound wild animals Amazon

Abstract in Portuguese:

ABSTRACT.- Alves F.R., Costa F.B., Arouche M.M.S., Barros A.C.E., Miglino M.A., Vulcano L.C. & Guerra P.C. 2007. [Ultrasonographic evaluation of the urinary system, liver and uterus of Cebus apella monkey.] Avaliação ultra-sonográfica do sistema urinário, fígado e útero do macaco-prego, Cebus apella. Pesquisa Veterinária Brasileira 27(9):377-382. Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP 05508-270, Brazil. E-mail: flaviovet@usp.br The Brown Capuchin, Cebus apella, has a wide distribution in the northern and southern Brazilian Amazon region and in the Cerrado (savanna). These monkeys are usually submitted to predatory chase, increasing the need for preservation of this wild animal species. An ultrasonographic examination of 10 Brown Capuchins was made in order to describe the normal ultrasonographic anatomy of their abdominal cavity. The urinary bladder revealed its wall thickness with an average of 0.2cm, the topographic situation of which allowed close relation with the wall of uterus and descendent colon. Using caudal abdominal scan, images of aorta, caudal vena cava and right iliac vein were obtained. Liver was accessible for examination by sagittal and cross-section ultrasound, allowing visualization of gallbladder and hepatic vessels. Renal scan allowed accuracy to evidence the echogenicity differences between pelvis, renal sinus, as well as the cortical-medullary relationship. The mean length of the kidneys was 6.24±0.31cm, and no significant differences were observed between left and right kidney length (Student’s t-test and ANOVA). The renal volume obtained was 2.37±0.18cm3. Correlation Coefficients of Pearson between right and left renal length and between right and left renal volume were r = 0.74 and 0.51. Mean thickness for cortical and medullar regions was 0.75±0.11 and 0.39±0.06cm, respectively. Correlation Coefficient of corticomedullar relation between right and left renal was r = 0.19. Examination by ultrasound was considered an efficient, non-invasive, fast and repeatable technique which provides useful data for clinicians and surgeons engaged in wild animal medicine.

Abstract in English:

ABSTRACT.- Alves F.R., Santos T.C., Freiberger S., Ambrósio C.E. & Miglino M.A. 2007. [The dynamic of precursor of the olfactory epithelium of mongrel dogs: an immuno-histochemical and ultrastructural study.] Dinâmica dos precursores celulares do epitélio olfatório de cães sem raça definida: um estudo imunohistoquímico e ultra-estrutural. Pesquisa Veterinária Brasileira 27(9): 388-392. Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, USP, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP 05508-270, Brazil. E-mail: flaviovet@usp.br Olfactory epithelium presents a mechanism of differentiation where stem cells give arise to amplifying progenitor cell which express Mammalian Achaete Scute Homolog 1 (Mash1). These cells can be differentiated into olfactory receptors. An immunolocalization study and ultrastructural analysis by transmission electron microscopy of olfactory epithelium of mongrel dogs were made using 3 males (one year old) and 2 females (three years old). Labeled cells with positive staining by Proliferating cell nuclear antigen (PCNA) were observed in specific areas of the olfactory epithelium, especially above the basal membrane. The ultrastructure revealed cells adjacent to the basal membrane with morphology resembling sustentacular cells, supporting the idea of renewal of sustentacular and olfactory sensorial cells. Olfactory epithelium contains basal cells committed to self-renewal, characterized by high metabolic activity, identified by positive reaction to PCNA. These results suggested the renewal of sustentacular and sensorial olfactory cells through the same pathway.

• Olfactory epithelium basal cells precursor cells PCNA method

Abstract in Portuguese:

ABSTRACT.- Alves F.R., Santos T.C., Freiberger S., Ambrósio C.E. & Miglino M.A. 2007. [The dynamic of precursor of the olfactory epithelium of mongrel dogs: an immuno-histochemical and ultrastructural study.] Dinâmica dos precursores celulares do epitélio olfatório de cães sem raça definida: um estudo imunohistoquímico e ultra-estrutural. Pesquisa Veterinária Brasileira 27(9): 388-392. Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, USP, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo, SP 05508-270, Brazil. E-mail: flaviovet@usp.br Olfactory epithelium presents a mechanism of differentiation where stem cells give arise to amplifying progenitor cell which express Mammalian Achaete Scute Homolog 1 (Mash1). These cells can be differentiated into olfactory receptors. An immunolocalization study and ultrastructural analysis by transmission electron microscopy of olfactory epithelium of mongrel dogs were made using 3 males (one year old) and 2 females (three years old). Labeled cells with positive staining by Proliferating cell nuclear antigen (PCNA) were observed in specific areas of the olfactory epithelium, especially above the basal membrane. The ultrastructure revealed cells adjacent to the basal membrane with morphology resembling sustentacular cells, supporting the idea of renewal of sustentacular and olfactory sensorial cells. Olfactory epithelium contains basal cells committed to self-renewal, characterized by high metabolic activity, identified by positive reaction to PCNA. These results suggested the renewal of sustentacular and sensorial olfactory cells through the same pathway.

Abstract in English:

ABSTRACT.- Inoe A.P., Pereira F.C., Stopiglia A.J. & Da-Silva C.F. 2007. Pharmacological immunomodulation enhances peripheral nerve regeneration. Pesquisa Veterinária Brasileira 27(9):363-369. Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Cidade Universitária, Av. Prof. Lineu Prestes 2415, São Paulo, SP 055508-900, Brazil. E-mail: ana.paula@unipar.br To assess the effect of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine MDP topically administrated on the regenerating peripheral neurons, twelve male C57BL/6J adult mice were equally distributed into three groups. Four mice underwent unilateral sciatic nerve transection and polyethylene tubulization, with a 4mm gap between the proximal and distal nerve stumps and were implanted with collagen + PBS (COL). Other four animals underwent the same surgical procedure but received collagen + MDP (COL/MDP) inside the prosthesis. Four animals were not operated and served as control group (NOR). After 4 weeks, the regenerated nerve cables were processed for total myelinated axon counting and myelinated fiber diameter measurement. The L5 dorsal root ganglion (DRG) was also removed and sectioned for sensory neurons counting and measurement. The results revealed significant difference (p<0.05) in axonal counting among the groups NOR (4,355±32), COL (1,869±289) and COL/MDP (2,430±223). There was a significant reduction in the axonal diameter in the operated groups (COL=3.38mm±1.16 and COL/MDP=3.54mm±1.16) compared to NOR (6.19mm±2.45). No difference was found in the number of DRG neurons between the experimental groups (COL=564±51; COL/MDP=514±56), which presented fewer sensory neurons compared to NOR (1,097±142). Data obtained indicate that locally applied MDP stimulates peripheral nerve regeneration in mice.

• N-Acetylmuramyl-L-Alanyl-D-Isoglutamine MDP nerve regeneration drug effect sciatic nerve surgery

Abstract in Portuguese:

ABSTRACT.- Inoe A.P., Pereira F.C., Stopiglia A.J. & Da-Silva C.F. 2007. Pharmacological immunomodulation enhances peripheral nerve regeneration. Pesquisa Veterinária Brasileira 27(9):363-369. Departamento de Anatomia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Cidade Universitária, Av. Prof. Lineu Prestes 2415, São Paulo, SP 055508-900, Brazil. E-mail: ana.paula@unipar.br To assess the effect of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine MDP topically administrated on the regenerating peripheral neurons, twelve male C57BL/6J adult mice were equally distributed into three groups. Four mice underwent unilateral sciatic nerve transection and polyethylene tubulization, with a 4mm gap between the proximal and distal nerve stumps and were implanted with collagen + PBS (COL). Other four animals underwent the same surgical procedure but received collagen + MDP (COL/MDP) inside the prosthesis. Four animals were not operated and served as control group (NOR). After 4 weeks, the regenerated nerve cables were processed for total myelinated axon counting and myelinated fiber diameter measurement. The L5 dorsal root ganglion (DRG) was also removed and sectioned for sensory neurons counting and measurement. The results revealed significant difference (p<0.05) in axonal counting among the groups NOR (4,355±32), COL (1,869±289) and COL/MDP (2,430±223). There was a significant reduction in the axonal diameter in the operated groups (COL=3.38mm±1.16 and COL/MDP=3.54mm±1.16) compared to NOR (6.19mm±2.45). No difference was found in the number of DRG neurons between the experimental groups (COL=564±51; COL/MDP=514±56), which presented fewer sensory neurons compared to NOR (1,097±142). Data obtained indicate that locally applied MDP stimulates peripheral nerve regeneration in mice.

Abstract in English:

ABSTRACT.- La Paz M.N., Fonseca V.U., Campos D.B., Artoni L.P., Sousa L.M.M.C. & Papa P.C. 2007. [In vitro progesterone production from bovine corpus luteum throughout gestation.] Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação. Pesquisa Veterinária Brasileira 27(9):370376. Setor de Anatomia, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques Paiva, 87, SP 05508-270, Brazil. E-mail: ppapa@usp.br The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20oC until processing. Progesterone was measured by RIA and protein content by Lowry’s method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern in vitro resembled what occurs in vivo.

• Corpus luteum gestation progesterone cell culture

Abstract in Portuguese:

ABSTRACT.- La Paz M.N., Fonseca V.U., Campos D.B., Artoni L.P., Sousa L.M.M.C. & Papa P.C. 2007. [In vitro progesterone production from bovine corpus luteum throughout gestation.] Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação. Pesquisa Veterinária Brasileira 27(9):370376. Setor de Anatomia, Departamento de Cirurgia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques Paiva, 87, SP 05508-270, Brazil. E-mail: ppapa@usp.br The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20oC until processing. Progesterone was measured by RIA and protein content by Lowry’s method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern in vitro resembled what occurs in vivo.