Medycyna Wet. 2006, 62 (5) 524
Praca oryginalna Original paper
In general, physiological changes in the mucous membrane of the reproductive tract during individual phases of the reproductive cycle reveal the level of regression of involution processes with the activity of hydrolytic enzymes of the epithelial cells being consi-dered one of the crucial indicators of these changes.
Depending on the phase of the estrous cycle many studies have confirmed pronounced functional chan-ges in the epithelial cells of the endometrium e.g. decreased alkaline phosphatase (AlP) activity in this tissue was noted in cows and heifers in late diestrus (15) with acidic phosphatase (AcP) activity peaking (13) in the same period. The highest values of AlP activity were observed in the epithelium of the endo-metrium of sows during estrus when compared with metestrus (12). A similar increase in AlP activity in the oviductal epithelial cells was found in sheep (11) and sows (4) during the estrous stage.
There was an intense reaction to non-specific ester-ase (NsE) in the surface epithelial cells and surface endometrial glands during the follicular phase. NsE
activity in non-ciliary oviductal cells peaked in estrus with some of its activity being retained until late pro-estrus (15).
In addition to these changes observed during pre-gnancy, Krajnièáková et al. (7) reported a significant increase in AlP in the oviductal epithelial cells from day 3 to day 40 in the postpartum period of goats without litter size being taken into account. A similar increase in AlP activity during the puerperal period mainly in ciliary cells of the oviductal epithelium was noted also in laboratory animals (3).
There is relatively a paucity of data on postpartum changes in the activity of these enzymes in ewes. Moreover, the above mentioned studies dealing with the activity of these enzymes in the reproductive tract during the reproductive cycle were focused neither on the dynamics of functional changes in the puerperal period of ewes nor on a possible effect of litter size on the length of puerperium in these animals. For this reason the aim of this experiment was to observe AlP, AcP and NsE activity in secretory and ciliary cells in the oviducts of puerperal ewes e.i. on days 7, 17, 25 and 35 a, in ewes with a singleton b, in ewes with twins.
Influence of litter size on histochemical picture
of ewe oviduct during the puerperal period*
)
IGOR VALOCKY, ¼UDOVÍT LENHARDT, TEFAN MOZE*
University of Veterinary Medicine, Komenského 73, 041 81 Koice, Slovak Republic
*Institute of Animal Physiology Slovak Academy of Sciences, oltesovej 4-6, Koice, Slovak Republic Valocký I., Lenhardt ¼., Moze .
Influence of litter size on the histochemical picture of ewe oviduct during the puerperal period Summary
The study focuses on the activity of alkaline phosphatase, acidic phosphatase and non-specific esterase in the epithelial cells of the oviducts of puerperal ewes with a single lamb (n = 24) and two lambs (n = 24). The experimental animals had the ampullar sections of the oviducts removed on days 7, 17, 25 and 35 postpartum. The results indicated an upward trend in the activity of the investigated enzymes in both groups of animals from day 7 to 35 of the puerperal period. However, the increase in enzyme activity in the ewes with twins was slower in comparison with the ewes with a singleton, which was significantly manifested by lower alkaline phosphatase activity (5.8 ± 0.4 vs 4.3 ± 0.4; P < 0.01), acidic phosphatase activity (11.5 ± 0.3 vs 7.2 ± 0.4; P < 0.05) and non--specific esterase activity (11.3 ± 0.4 vs 8.7 ± 0.4; P < 0.05) on day 25 postpartum. These differences between the groups persisted until the completion of the experiment with alkaline phosphatase activity (7.1 ± 0.7 vs 5.7 ± 0.3; P < 0.05), acidic phosphatase activity (14.4 ± 0.4 vs 8.6 ± 0.3; P < 0.001) and non-specific esterase activity (11.6 ± 0.5 vs 9.5 ± 0.4; P < 0.01) on day 35 postpartum.
The results indicate that the retardation of functional changes in the ewes with twins reveals the prolongation of time essential for the completion of regenerative processes in the epithelium of the oviducts. The discussion deals with the possible utilization of these facts in relation to optimizing out of season breeding in sheep.
Keywords: alkaline phosphatase, acidic phosphatase, non-specific esterase, oviduct
*) Acknowledgments: These studies were funded by Ministry of Education
Medycyna Wet. 2006, 62 (5) 525
Material and methods
The experiment was conducted at the accreditation of Ethical committee at the University of Veterinary Medici-ne in Koice (5, 6).
Experimental animals. The experiment was conducted on 48 ewes, 3-4 years old of the Slovak merino breed (mean body weight 38.5 ± 1.9 kg) divided into two groups: a) ewes with a single lamb (n = 24); b) ewes with twins (n = 24). During the experiment the animals were kept individually in cotes and had a free access to feed and drinking water. Feed ration per animal/day consisted of meadow hay, feed concentrate, and root crops. A mineral supplement (Se, Zn, J, Cu, Co, Mg) and water were availa-ble ad libitum (14).
The ewes from both groups (n = 6 per day) were eutha-nised (Thiopental ® inj., Léèivá CZ) on days 7, 17, 25 and 35 postpartum (p.p.) according to an advance schedule. After bleeding the excised samples from the ampullar sec-tion of oviducts were immediately frozen in liquid nitro-gen vapour and stored in a freezer at 20° until processed. Enzyme assay. The demonstration of AlP activity was performed by using the modified simultaneous azocoupling method (9) with naphthol AS-BI phosphate (Sigma, Deisenhofen, Germany) and Fast Blue BB (Sigma). AcP activity was established by using the simultaneous azo--coupling method (9) with naphtol-AS-MX-phosphate (Fluka, Germany) and hexazo-p-rozaniline (Serva, Germa-ny) and NsE activity was established by using naphtol-AS--D-acetate (Fluka, Germany) and ferrocyanide (9).
Enzyme activity was cytophotometrically analyzed with a Vickers M85 microdensitometer. The measurements were performed by using an × 40 objective, an effective scan-ning area of 28.3 µm3 and a scanning spot of 0.5 µm. The
integrated absorbance was measured at a wavelength of 480 nm for AlP and 540 nm for AcP and NsE. The mask was set over in at least 30 areas of oviduct in five tissue sections. The activity of enzymes was calculated as the absorbance values recorded by the instrument/min./µm3
of oviductal cells ± S.E.M. and these mean values were referred to one animal.
Statistical evaluation. Statistical evaluation of the results was performed by one-way analysis of variance (ANOVA). Significance of differences between means was calculated by Tukeys test. The two-way ANOVA was used to test the effect of the number of lambs on the activity of
enzymes during puerperium and their interaction. Sigma Stat statistical software (Jandel Scientific 2000) was utilized for the analysis of data.
Results and discussion
The evaluation of AlP, AcP and NsE activity in the cells of the epithelium of ewe oviducts at individual time intervals of puerperium is set out in tab. 1. The group of ewes with a singleton exhibited a statistically significant increase in AlP activity in the oviductal epithelial cells on day 25 and 35 postpartum (P < 0.01) when compared with day 7 postpartum.
It was an increase of about 40 percent on day 25 and about 50 percent on day 35. A statistically significant increase in AlP activity in the group of ewes with twins was observed only on day 35 (P < 0.01). This increase on day 35 compared with day 7 represented about 50 percent. The evaluation of AlP activity depending on litter size exhibited markedly lower values in the ewes with twins on days 25 and 35 postpartum (P < 0.01, P < 0.05 respectively). Two-way ANOVA confirmed a specific effect of litter size on AlP activity during puerperium (interaction F = 9.18; P < 0.001).
The evaluation of AcP localized in cytoplasm of cylindrical cells in the oviduct of the ewes with a sin-gleton revealed a significant increase in its activity. In contrast with day 7 an increase of 25 percent was observed on day 17 (P < 0.01), an increase of about 50 percent on day 25 (P < 0.001) and an increase of about 60 percent on day 35 (P < 0.001). When evaluating AcP activity in the group of ewes with twin lambs, a significant increase (P < 0.01) was noted on day 25 (about 30 percent) and this upward trend was mainta-ined also on day 35 postpartum (about 40 percent). The group with a single lamb and that with twins significantly differed between themselves on day 25 (P < 0.01) and day 35 (P < 0.001). A specific influence of litter size on AcP activity during the puerperal pe-riod was confirmed by two-way ANOVA (interaction F = 8.93; (P < 0.001).
Similar quantitative changes as in AcP were noted also in NsE. NsE activity in the oviductal epithelium of the ewes with a singleton exhibited a significant increase on days 17, 25 and 35 (P < 0.05, resp. P < 0.01),
m u tr a p t s o P s y a D Alkailnephosphatase Acidicphosphatase Non-speciifcesterase S w E EwT EwS EwT EwS EwT 7 3.436±0.422 2.770±0.538 5.264±0.382 4.833±0.319 7.921±0.426 6.835±0.382 7 1 3.754±0.429 3.905±0.453 7.042b±0.275 5.295±0.417 9.875a±0.372 7.526±0.293 5 2 5.868b±0.382 4.260±0.408** 11.537c±0.329 7.211b**±0.423 11.277b±0.355 8.714a*±0.442 5 3 7.094b±0.679 5.651±0.326b* 14.382c±0.420 8.573b***±0.259 11.564b±0.461 9.523b**±0.387
Tab. 1. Mean (± S.E.M.) values of AlP, AcP and NsE activity in the oviduct of ewes with a singleton and twins during puerperium
Explanation: EwS ewes with singleton, EwT ewes with twins; the data are means ± S.E.M. AlP, AcP and NsE activity is given as the
integrated absorbance in min./µm3 brush border of the epithelial cells of apical parts of the oviducts at a wavelength of 480 and 520 nm.
Significantly different from the single lamb group: * P < 0.05, ** P < 0.01, *** P < 0.001. Significantly different from day 7 groups: a, P < 0.05; b, P < 0.01; c, P < 0.001
Medycyna Wet. 2006, 62 (5) 526
which represented about 20-30 percent compared with day 7. A significant increase in NsE activity in the ewes with twins occurred on day 25 (P < 0.05 about 20 percent) and this upward trend was maintained also on day 35 postpartum (P < 0.01 about 30%). The values of NsE activity were significantly lower on day 25 (P < 0.05) and day 35 (P < 0.01) in the group with twins when compared with the group with a singleton. A specific effect of litter size on NsE activity during puerperium was confirmed by two-way ANOVA (interaction F = 8.86; P < 0.01).
The results obtained in this experiment exhibited an upward trend of the activity of individual enzymes localized in the epithelial cells of apical parts of the oviducts in both groups of animals from day 7 to day 35 of the puerperal period. However, the intensity of these changes in the ewes with twins was lower than that in the ewes with a singleton, which is indicated by significant differences in AlP, AcP and NsE activity on days 25 and 35 within the framework of these groups. The changes in AlP activity in the sheep in this expe-riment were to a certain extent consistent with literary data which referred to a similar tendency in goats from day 3 to day 40 of the puerperal period i.e. With the progressing postpartum period AlP activity increased in the ciliary cells of oviductal epithelium in these ani-mals (7). As claimed by Clark (3) changes in AlP acti-vity are closely related to transphosphorylation mecha-nisms involved in energy transformation necessary for kinocilia movement. From this point of view, the gra-dual increase in AlP activity in these observations can be related to the preparation of the reproductive tract for a new reproductive cycle. The completion of repa-rative processes in the uterus and oviducts in ewes with a singleton occurs in Central Europe by day 34 post-partum (8). In this context the time necessary for the completion of reparative processes in this experiment with the ewes with twins was not sufficient enough (35 days) for enzymatic processes of AlP activity in the oviductal epithelial cells to reach a complete rege-neration in comparison with the ewes with a single-ton. In comparison with AlP AcP activity exhibited a more pronounced retardation of its increase in the ewes with twins.
NsE activity in the oviductal epithelial cells ob-served in both groups of sheep showed a continual upward trend at the observed intervals. Activity simi-lar to that observed on day 35 postpartum in the ewes with a single lamb was reported in goats during estrus by Bhattacharya and Saigal (1). The acceleration of activity of this enzyme (similarly as that of AcP) was slower in the group of ewes with twins when compa-red with the ewes with a singleton. An increase in the number of secretory cells and the presence of nume-rous kinocilia in the ciliary cells is typical of prolifera-tion processes occurring during puerperium. The num-ber of secretory cells exhibits a significant increase from day 3 to day 40 postpartum (2, 10). From this
point of view, the results of activity changes of NsE observed in this experiment indicate their possible in-volvement in energy transformation processes essen-tial for the fertilization process following the postpar-tum period.
The evaluation of results reveals a significant retar-dation of activity of the observed tissue enzymes in the oviducts during the puerperal period in the ewes with twins compared with the ewes with a singleton. It is highly probable that the regenerative processes and a longer period of time necessary for their optimal adjustment in the ewes with twins demonstrate the re-tardation of functional changes related to the transfor-mation of mobilizable energy in the epithelium of the oviduct. The question remains whether the altered en-zyme activity resulting from different litter size may play a key role in puerperal management. Extension of knowledge of the whole of puerperium until the first estrous cycle would enable both analysis and evalu-ation of these processes in relevalu-ation to optimizevalu-ation of out of season breeding in sheep.
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