Structural and Morphometric changes in Ruminal tissues and physiological effects in Wethers with plastic bag-impacted Rumen

Abstract

Freely grazing ruminants are liable to both advertent and accidental ingestion of indigestible foreign materials. Rumen impaction with such materials induces stress, damages ruminal tissues and impairs ruminal functional efficiency. Quantitative data on surface area and volume depicting structural changes in ruminal tissues consequent to impaction with plastic bags is lacking. Furthermore, the physiological effects resulting from impaction have not been documented. The objectives of this study include: 1. To investigate the quantitative structural changes in ruminal tissues of wethers following experimental impaction of the rumen with plastic bags. 2. To analyze the levels of cortisol in plasma of wethers implanted with plastic bags in their rumen as an indicator of stress. 3. To estimate faecal cortisol metabolite levels as an indicator of stress in wethers with plastic bags implanted in their rumen. The experimental set-up was in two Phases: Phase I of the experiment had a 4-week endpoint and Phase II an 8-week endpoint after implanting the rumen with plastic bags. Each phase had 3 groups of wethers consisting of 5 animals per group. The groups in Phase I were designated as test group (PPI), positive control group (NPPI) and negative control group (CPI) all of which were sacrificed at 4 weeks. Phase II groups were designated as PPII, NPPII and CPII for test, positive control and negative control groups respectively and were sacrificed at 8 weeks. The groups PPI and PPII were implanted with 166g of plastic bags in the rumen through rumenotomy. Groups NPPI and NPPII underwent rumenotomy but no plastic bags were implanted. The groups CPI and CPII had neither rumenotomy nor plastic bags. Following euthanasia, the rumen from each animal was dissected out and divided into the various sacs for gross examination, macroscopic surface area measurements by point-associated area method and reference volume of each sac was determined by Scherle’s method. Tissue blocks for histology were obtained by systematic random sampling and processed to obtain vertical sections for surface and volume density estimations. At the microscopic level, STEPanizer® stereological software was employed to estimate volume and surface densities of each ruminal sac and subsequently absolute values were calculated. Short-term (the first 72 hour period) and long-term (8-week period) plasma cortisol levels were determined for each wether, using enzyme-linked immunosorbent assay (ELISA) to indicate presence or absence of stress. Faecal cortisol metabolite levels were analyzed for each wether as an enhanced method for estimation of stress, using an 11-oxoaetiocholanolone enzyme immuno-assay (EIA I) kit. The test groups PPI and PPII with rumen impaction showed severe histological changes particularly in the mucosal wall of the rumen compared to wethers without impaction. The papillae observed in the test group of wethers were stunted, bent over, slender and constricted and this was more severe in the cranial and ventral sacs, with the extent of severity being correlated to the duration of impaction. Stereological analysis of the entire rumen from normal wethers in CPII indicated a total mean macroscopic surface area of 0.109m2. Microscopically, the absolute mean absorptive surface area of the entire rumen was 0.473 ± 0.017m2 of which the ventral sac (VS) was the largest at 0.186 ± 0.010m2, then cranial sac AR at 0.101 ± 0.010m2, the dorsal sac (DS) at 0.090 ± 0.007m2, the caudoventral blind sac (CVB) at, 0.079 ± 0.007m2 and the smallest was the caudodorsal blind sac (CDB) at 0.016 ± 0.001m2. The total mean absolute volume of ruminal tissues was 538cm3 of which the mucosa, submucosa, muscularis interna, muscularis externa and serosa were 162cm3, 107cm3, 158cm3, 98cm3 and 13cm3 respectively. Stereological evaluations revealed that the rumen impacted with plastic bags had severe loss in surface area and volume estimates of the absorptive mucosa, which was more evident in Phase II wethers (PPII). The mean surface area of AR, DS, VS and CVB in PPII wethers decreased by 50.8% (p < 0.0001), 29.6% (p = 0.0227), 41.1% (p < 0.0001) and 37.5% (p = 0.0227) respectively when compared to the corresponding ruminal sacs from NPPII. The mean absolute volume of mucosae in AR, DS, VS, CDB and CVB significantly reduced (52.4%, p < 0.0001; 55.1%, p = 0.0006; 32.3%, p = 0.0427; 58.8%, p = 0.0004 and 55.0%, p = 0.0002 respectively) compared to the corresponding sacs in NPPII. At 8 weeks post-impaction the total mean surface area and volume of mucosal tissue in the entire rumen from PPII reduced significantly (39.7%, p < 0.0001; 46.9%, p = 0.0004 respectively) compared to rumen from NPPII. The total mean surface area of the rumen from wethers in PPII, NPPII and CPII were 0.282 ± 0.012m2, 0.467 ± 0.017m2 and 0.473 ± 0.017m2 respectively. The mean absolute volume of mucosae in the entire rumen of PPII, NPPII and CPII were 82.7cm3, 155.82cm3 and 162.48cm3 respectively. Thus absolute volume of mucosa in the rumen of wethers in PPII formed 17% of their entire rumen volume, while that of either NPPII or CPII formed 30% of their entire rumen volume. A lower value in body-mass-standardized total surface area of 0.0113m2kg-1 was recorded in PPII compared to 0.0163m2kg-1 obtained for NPPII. A lower value of 3.31cm3kg-1 in body-massstandardized total volume of rumen mucosa was obtained for rumen from wethers in PPII compared to 5.45cm3kg-1 recorded in NPPII. Plasma cortisol concentration significantly (p < 0.05) increased about 4-fold during the first 72 hours in wethers whose rumen were implanted with plastic bags. The effect of interaction between the experimental groups and time was also significant (p = 0.0420). The elevated levels in plasma cortisol persisted over the next three weeks then gradually declined over the remaining 5-week period that followed. Consequently, at the end of 8-week post-implantation, mean plasma cortisol levels had reverted to normal range. However, there was significant (p = 0.0070) interaction between the different groups with time. The concentration of faecal cortisol metabolites in the test group PPI significantly increased over 5-fold (p = 0.0023) to 3-fold (p = 0.0294) from baseline values during the initial 24 hour and 72 hour period post-impaction respectively. This rise in FCM levels persisted over the next 4 weeks as well as in the 6th week but levels declined to normal by the 8th week of experimentation. There were no significant differences in gross, histological and stereological parameters between the positive and negative control groups. However, the positive control group had about 1.5 times increased plasma cortisol concentration 6 hours after rumenotomy, but decreased to normal levels by 72 hours. From the current study it was concluded that prolonged impaction of the rumen of wethers with indigestible plastic bags is likely to cause the following outcomes which might subsequently diminish ruminal functional efficiency and loss of health in the animal: a) Severe loss in body weights of wethers. b) Severe histological changes in the ruminal mucosae of wethers. c) Progressive loss in structural quantities in the ruminal wall the extent of which is related to the duration of impaction. d) Severe damage of the absorptive surface area and loss in volume of tissues in the rumen particularly mucosal volume may impair rumen function consequently affecting body weight in ruminants. e) Increased levels of plasma cortisol and faecal cortisol metabolite which is more acute than chronic is indicative of stress in rumen impaction. Therefore ingestion of waste plastic bags by small ruminants should not be underrated, because it gradually leads to rumen impaction that induces stress and loss of body weight with subsequent structural changes in ruminal tissues that overall leads to loss of health in the animal. Creating public awareness and educating the famer on the devastating effect of ingestion of plastic bags on ruminant’s health and the proper disposal of waste plastic bags are recommended. This will improve productivity and help the farmer get better returns.